Polynucleotide primers and probes

ABSTRACT

The present invention provides a novel technology that involves improved primer design. These primer pairs have a wide range of applications and provide high sensitivity and specificity.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/092,693, filed Nov. 27, 2013, now U.S. Pat. No. 10,480,030, which isa continuation of U.S. patent application Ser. No. 12/913,742, filedOct. 27, 2010, which claims priority to U.S. Provisional PatentApplication No. 61/255,461, filed Oct. 27, 2009, the disclosure of whichare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to polynucleotide combinations and their use.

BACKGROUND

Detection and amplification of nucleic acids play important roles ingenetic analysis, molecular diagnostics, and drug discovery. Many suchapplications require specific, sensitive and inexpensive quantitativedetection of certain DNA or RNA molecules, gene expression, DNAmutations or DNA methylation present in a small fraction of totalpolynucleotides. Many current methods use polymerase chain reaction, orPCR, and specifically, real-time PCR (quantitative, or qPCR) to detectand quantify very small amounts of DNA or RNA from clinical samples.

While the performance of current PCR assays is constantly improving,their sensitivity, specificity and cost are still far away from becominga widely acceptable diagnostic test. Indeed, many PCR methods currentlyused in the art suffer from technical limitations that make the methodsinadequate for many practical applications. For example, in instanceswhere the target molecule has secondary structure that inhibits or evencompletely prevents binding of one or both primers to the target,amplification can be reduced or even non-existent, which, for example,from a diagnostic standpoint could give rise to a false negative despiteuse of a highly specific primer with binding properties that would beexpected to be sensitive. Other challenges include low sensitivity ofcurrent real-time PCR assays in detection and discrimination of rare DNAmolecules with a single base mutation in situations when they mixed withthousands of non-mutated DNA molecules, and ability to combine multiplemutation detection assays into one multiplex diagnostic assay.

There thus remains a need in the art for a development of amplificationprimers that combines high binding specificity with low synthesis costthat retain the ability to overcome technical problems recognized in theart, including novel application of PCR for diagnostics using nextgeneration sequencing platforms.

SUMMARY OF THE INVENTION

In one aspect, the disclosure provides a polynucleotide primercombination comprising a first polynucleotide and a secondpolynucleotide, wherein the first polynucleotide (P) comprises a firstdomain (Pa) having a sequence that is complementary to a first targetpolynucleotide region (T₁) and a second domain (Pc) comprises a uniquepolynucleotide sequence, and the second polynucleotide (F) comprises afirst domain (Fb) having a sequence that is complementary to a secondtarget polynucleotide region (T₂) and a second domain (Fd) comprising apolynucleotide sequence sufficiently complementary to Pc such that Pcand Fd will hybridize under appropriate conditions, and wherein thetarget polynucleotide has a secondary structure that is denatured byhybridization of Fb to the target polynucleotide. In one aspect, thesecondary structure of the target polynucleotide inhibits polymeraseextension of the target polynucleotide in the absence of F. Thedisclosure further contemplates an aspect wherein the polynucleotideprimer combination P and/or F further comprise a modified nucleic acid.

The disclosure further provides a polynucleotide primer combinationcomprising a first polynucleotide and a second polynucleotide, whereinthe first polynucleotide (P) comprises a first domain (Pa) having asequence that is complementary to a first target polynucleotide region(T₁) and a second domain (Pc) comprising a unique polynucleotidesequence, and the second polynucleotide (F) comprises a first domain(Fb) having a sequence that is complementary to a second targetpolynucleotide region (T₂) and a second domain (Fd) comprising apolynucleotide sequence sufficiently complementary to Pc such that Pcand Fd will hybridize under appropriate conditions, and wherein P and/orF further comprise a modified nucleic acid.

A polynucleotide primer combination is also provided comprising a firstpolynucleotide, a second polynucleotide, and a blocker polynucleotide,the first polynucleotide (P) comprising a first domain (Pa) having asequence that is complementary to a first target polynucleotide region(T₁) and a second domain (Pc) comprising a unique polynucleotidesequence, the second polynucleotide (F) comprising a first domain (Fb)having a sequence that is complementary to a second targetpolynucleotide region (T₂) and a second domain (Fd) comprising apolynucleotide sequence sufficiently complementary to Pc such that Pcand Fd will hybridize under appropriate conditions, and the blockerpolynucleotide comprising a nucleotide sequence that is complementary toa third target polynucleotide region (T₃), wherein T₃ is located 5′ ofT₁ and T₂. In an aspect of the primer combination, a nucleotide at the3′ end of P and a nucleotide at the 5′ end of the blocker polynucleotideoverlap. In another aspect, the blocker polynucleotide has a sequencethat overlaps Pa over the whole length of Pa. In still another aspect,the nucleotide at the 3′ end of P and the nucleotide at the 5′ end ofthe blocker polynucleotide are different. In each of these aspects, anembodiment is contemplated wherein P, F, and/or the blockerpolynucleotide comprises a modified nucleic acid.

The disclosure further provides a polynucleotide primer combinationcomprising a first polynucleotide, a second polynucleotide, and a probepolynucleotide, the first polynucleotide comprising a first domain (Pa)that is complementary to a first target polynucleotide region (T₁) and asecond domain (Pc) comprising a unique polynucleotide sequence, thesecond polynucleotide (F) comprising a first domain (Fb) that iscomplementary to a second target polynucleotide region (T₂) and a seconddomain (Fd) comprising a polynucleotide sequence sufficientlycomplementary to Pc such that Pc and Fd will hybridize under appropriateconditions, and the probe polynucleotide comprising a nucleotidesequence that is complementary to a third target polynucleotide region(T₄), wherein T₄ is located 5′ of T₁ and T₂. In certain aspects, theprobe polynucleotide comprises a label and a quencher. In other aspects,P, F and/or the probe polynucleotide comprise a modified nucleic acid.An embodiment is also provide wherein the polynucleotide primercombination further comprises a blocker polynucleotide, wherein theblocker polynucleotide comprises a nucleotide sequence that iscomplementary to a fourth target polynucleotide region (T₃), and whereinT₃ is located 5′ of T₁ and T₂ and 3′ of T₄. In one aspect, the blockercomprises a modified nucleic acid.

Also provided is a polynucleotide primer combination comprising a firstpolynucleotide, a second polynucleotide, and a universal quencherpolynucleotide, the first polynucleotide (P) comprising a first domain(Pa) that is complementary to a first target polynucleotide region (T₁),a second domain (Pc) comprising a unique polynucleotide sequence, and alabel at its 5′ end, the second polynucleotide (F) comprising a firstdomain (Fb) that is complementary to a second target polynucleotideregion (T₂) and a second domain (Fd) comprising two polynucleotidesequences, a 5′ polynucleotide sequence that is sufficientlycomplementary to the 5′ sequence of Pc such that the 5′ polynucleotidesequence of Pc and Fd will hybridize under appropriate conditions, and a3′ polynucleotide sequence that is sufficiently complementary to theuniversal quencher polynucleotide such that the 3′ polynucleotidesequence of Fd and the universal quencher will hybridize underappropriate conditions, and the universal quencher polynucleotidecomprising a quencher and a nucleotide sequence that is sufficientlycomplementary to the 3′ polynucleotide sequence of Fd such that theuniversal quencher polynucleotide and the 3′ polynucleotide sequence ofFd will hybridize under appropriate conditions. In one aspect, P, Fand/or the universal quencher polynucleotide comprise a modified nucleicacid. In another aspect, the polynucleotide primer combination furthercomprises a reverse primer, wherein the reverse primer comprises apolynucleotide sequence complementary to a polynucleotide strandcomprising a sequence that hybridizes to T₁.

In various aspects of any of the primer combinations provided herein, Pcomprises a modified nucleic acid. In other aspects, F further comprisesa modified nucleic acid, and in certain of these aspects, the modifiednucleic acid is in Pa, and/or the modified nucleic acid is in

Fb.

In each polynucleotide primer combination of the disclosure, as aspectis provided wherein P comprises a plurality of modified nucleic acids inPa, and/or wherein F comprises a plurality of modified nucleic acids inFb. When P comprises a modified nucleic acid, as aspect is providedwherein the modified nucleic acid is the nucleotide at a 3′ end of P.

In each primer combination disclosed, a aspects are provided wherein Fdis at least 70% complementary to Pc, wherein Pc is at least 70%complementary to Fd, wherein Pc and Fd hybridize to each other in theabsence of the template polynucleotide, wherein P is DNA, modified DNA,RNA, modified RNA, peptide nucleic acid (PNA), or combinations thereof,and/or wherein F is DNA, modified DNA, RNA, modified RNA, peptidenucleic acid (PNA), or combinations thereof.

In each primer pair combination, an aspect is provided wherein thepolynucleotide primer combination further comprises a blocking groupattached to F at its 3′ end which blocks extension from a DNApolymerase. In this aspect, an embodiment is provided wherein theblocking group is selected from the group consisting of a 3′ phosphategroup, a 3′ amino group, a dideoxy nucleotide, and an inverteddeoxythymidine (dT).

In another aspect of each primer pair combinations, certain embodimentsare provided wherein Pa is from about 5 bases in length to about 30bases in length, about 5 bases in length to about 20 bases in length,about 5 bases in length to about 15 bases in length, about 5 bases inlength to about 10 bases in length, about 5 bases in length to about 8bases in length. In other aspects, Pc is from about 5 bases in length toabout 200 bases in length, about 5 bases in length to about 150 bases inlength, about 5 bases in length to about 100 bases in length, about 5bases in length to about 50 bases in length, about 5 bases in length toabout 45 bases in length, about 5 bases in length to about 40 bases inlength, about 5 bases in length to about 35 bases in length, about 5bases in length to about 30 bases in length, about 5 bases in length toabout 25 bases in length, about 5 bases in length to about 20 bases inlength, about 5 bases in length to about 15 bases in length, about 10 toabout 50 bases in length, about 10 bases in length to about 45 bases inlength, about 10 bases in length to about 40 bases in length, about 10bases in length to about 35 bases in length, about 10 bases in length toabout 30 bases in length, about 10 bases in length to about 25 bases inlength, about 10 bases in length to about 20 bases in length, or about10 bases in length to about 15 bases in length. In still other aspects,Fb is from about 10 bases in length to about 5000 bases in length, about10 bases in length to about 4000 bases in length, about 10 bases inlength to about 3000 bases in length, about 10 bases in length to about2000 bases in length, about 10 bases in length to about 1000 bases inlength, about 10 bases in length to about 500 bases in length, about 10bases in length to about 250 bases in length, about 10 bases in lengthto about 200 bases in length, about 10 bases in length to about 150bases in length, about 10 bases in length to about 100 bases in length,about 10 bases in length to about 95 bases in length, about 10 bases inlength to about 90 bases in length, about 10 bases in length to about 85bases in length, about 10 bases in length to about 80 bases in length,about 10 bases in length to about 75 bases in length, about 10 bases inlength to about 70 bases in length, about 10 bases in length to about 65bases in length, about 10 bases in length to about 60 bases in length,about 10 bases in length to about 55 bases in length, about 10 bases inlength to about 50 bases in length, about 10 bases in length to about 45bases in length, about 10 bases in length to about 40 bases in length,about 10 bases in length to about 35 bases in length, about 10 bases inlength to about 30 bases in length, or about 10 bases in length to about100 bases in length. In yet other aspects, Fd is from about 5 bases inlength to about 200 bases in length, about 5 bases in length to about150 bases in length, about 5 bases in length to about 100 bases inlength, about 5 bases in length to about 50 bases in length, about 5bases in length to about 45 bases in length, about 5 bases in length toabout 40 bases in length, about 5 bases in length to about 35 bases inlength, about 5 bases in length to about 30 bases in length, about 5bases in length to about 25 bases in length, about 5 bases in length toabout 20 bases in length, about 5 bases in length to about 15 bases inlength, about 10 to about 50 bases in length, about 10 bases in lengthto about 45 bases in length, about 10 bases in length to about 40 basesin length, about 10 bases in length to about 35 bases in length, about10 bases in length to about 30 bases in length, about 10 bases in lengthto about 25 bases in length, about 10 bases in length to about 20 basesin length, or about 10 bases in length to about 15 bases in length.

In each aspect of the primer pair combination provide, an embodimentincludes that wherein P comprises a label. In some aspects, the label islocated in P at its 5′ end and/or the label is quenchable. In someaspects of these embodiments, F comprises a quencher and/or the quencheris located in F at its 3′ end. In specific embodiments, the quencher isselected from the group consisting of Black Hole Quencher 1, Black HoleQuencher-2, Iowa Black FQ, Iowa Black RQ, and Dabcyl. G-base.

In certain primer pair combinations comprising a modified nucleic acid,the modified nucleic acid in the blocker polynucleotide is thenucleotide at the 5′ end of the blocker polynucleotide, the modifiednucleic acid is the nucleotide at the 3′ end of P, and/or the modifiednucleic acid is a locked nucleic acid.

The disclosure further provides a method of detecting the presence of atarget polynucleotide in a sample with a primer combination, the primercombination comprising a first polynucleotide and a secondpolynucleotide, the first polynucleotide (P) comprising a first domain(Pa) having a sequence that is fully complementary to a first targetpolynucleotide region (T₁) and a second domain (Pc) comprising a uniquepolynucleotide sequence, Pa having a sequence that is not fullycomplementary to a non-target polynucleotide in the sample and thesecond polynucleotide (F) comprising a first domain (Fb) that iscomplementary to a second target polynucleotide region (T₂) and a seconddomain (Fd) comprising a polynucleotide sequence sufficientlycomplementary to Pc such that Pc and Fd will hybridize under appropriateconditions, the method comprising the steps of: contacting the samplewith the primer combination and a polymerase under conditions that allowextension of a sequence from Pa which is complementary to the targetpolynucleotide when the target polynucleotide is present in the sampleand detecting the sequence extended from Pa indicating the presence ofthe target polynucleotide in the sample. In certain aspects, the methodprovides a change in sequence detection from a sample with a non-targetpolynucleotide compared to sequence detection from a sample with atarget polynucleotide.

Also provided is a method of detecting the presence of a targetpolynucleotide in a sample with a primer combination as disclosed hereinwherein P comprises a first domain that is fully complementary to T₁ andwherein Pa is not fully complementary to a non-target polynucleotide inthe sample, the method comprising the steps of: contacting the samplewith the primer combination and a polymerase under conditions that allowextension of a sequence from Pa which is complementary to the targetpolynucleotide when the target polynucleotide is present in the sampleand detecting the sequence extended from Pa, wherein detection indicatesthe presence of the target polynucleotide in the sample. In someaspects, the method provides a change in sequence detection from asample with a non-target polynucleotide compared to sequence detectionfrom a sample with a target polynucleotide.

In each of these methods, an embodiment is provided wherein thedetecting step is carried out using polymerase chain reaction. In theseembodiments, aspects are provided wherein the polymerase chain reactionutilizes P of the primer combination and a reverse primer, the reverseprimer having a sequence complementary to the sequence extended from Paand/or the polymerase chain reaction utilizes a reverse primercomplementary to the sequence extended from Pa and a forward primerhaving a sequence complementary to the strand of the targetpolynucleotide to which Pa hybridizes.

On another aspect of these methods, detection is carried out in realtime.

The disclosure further provides a method of initiating polymeraseextension on a target polynucleotide in a sample using a primercombination, the primer combination comprising a first polynucleotideand a second polynucleotide, the first polynucleotide (P) comprising afirst domain (Pa) having a sequence that is fully complementary to afirst target polynucleotide region (T₁) and a second domain (Pc)comprising a unique polynucleotide sequence, Pa having a sequence thatis not fully complementary to a non-target polynucleotide in the sampleand the second polynucleotide (F) comprising a first domain (Fb) that iscomplementary to a second target polynucleotide region (T₂) and a seconddomain (Fd) comprising a polynucleotide sequence sufficientlycomplementary to Pc such that Pc and Fd will hybridize under appropriateconditions, wherein the sample comprises a mixture of (i) a targetpolynucleotide that has a sequence (T₁) in a first region that is fullycomplementary to the sequence in Pa and (ii) a non-target polynucleotidethat has a sequence (T₁*) in a first region that is not fullycomplementary to Pa, the method comprising the step of contacting thesample with the primer combination and a polymerase under conditionsthat allow extension of a sequence from Pa and complementary to thetarget polynucleotide strand when Pa contacts T₁. In certain aspects ofthe method, the sequence in the first region (T₁) in the targetpolynucleotide differs from the sequence in the first region (T₁*) inthe non-target polynucleotide at one base. In other aspect, the methodfurther comprises the step of detecting the sequence extended from Pa,wherein detection indicates the presence of the target polynucleotide inthe sample.

The disclosure also provides a method of initiating polymerase extensionon a target polynucleotide in a sample using a primer combination asdisclosed herein, wherein P comprises a first domain (Pa) that is fullycomplementary to a first target polynucleotide region (T₁) and whereinPa is not fully complementary to a non-target polynucleotide in thesample, the method comprising the steps of: contacting the sample withthe primer combination and a polymerase under conditions that allowextension of a sequence from Pa which is complementary to the targetpolynucleotide when the target polynucleotide is present in the sample.In some aspects, the method further comprises the step of detecting thesequence extended from Pa, indicating the presence of the targetpolynucleotide in the sample. In various embodiments, the detecting stepis carried out using polymerase chain reaction, and in certain aspectsof this embodiment, the polymerase chain reaction utilizes P of theprimer combination and a reverse primer, the reverse primer having asequence complementary to the sequence extended from Pa, and/or thepolymerase chain reaction utilizes a reverse primer complementary to thesequence extended from Pa and a forward primer having a sequencecomplementary to the strand of the target polynucleotide to which Pahybridizes. In each embodiment of the method, an aspect is providedwherein detecting is carried out in real time.

Also provided is a method of amplifying a target polynucleotide in asample using a polynucleotide primer combination, the primer combinationcomprising a first polynucleotide and a second polynucleotide, the firstpolynucleotide (P) comprising a first domain (Pa) having a sequence thatis fully complementary to a first target polynucleotide region (T₁) anda second domain (Pc) comprising a unique polynucleotide sequence, Pahaving a sequence that is not fully complementary to a non-targetpolynucleotide in the sample and the second polynucleotide (F)comprising a first domain (Fb) that is complementary to a second targetpolynucleotide region (T₂) and a second domain (Fd) comprising apolynucleotide sequence sufficiently complementary to Pc such that Pcand Fd will hybridize under appropriate conditions, wherein the samplecomprises a mixture of (i) a target polynucleotide that has a sequencein a first region (T₁) that is fully complementary to the sequence in Paand (ii) one or more non-target polynucleotides that are not fullycomplementary to Pa; the method comprising the steps of: (a) contactingthe sample with the primer combination and a polymerase under conditionsthat allow extension of a sequence from Pa which is complementary to thetarget polynucleotide when the target polynucleotide is present in thesample, (b) denaturing the sequence extended from Pa from the targetpolynucleotide, and (c) repeating step (a) in the presence of a reverseprimer having a sequence complementary to a region in the sequenceextended from Pa in step (b) to amplify the target polynucleotide,wherein extension and amplification of the target polynucleotide occurswhen Pa is fully complementary to the sequence in the Pa but is lessefficient or does not occur when the first region in the targetpolynucleotide is not fully complementary to the sequence in Pa.

The disclosure also provides a method of amplifying a targetpolynucleotide in a sample using a polynucleotide primer combination asdisclosed herein, wherein the first polynucleotide (P) comprises a firstdomain (Pa) that is fully complementary to a first target polynucleotideregion (T₁) and wherein Pa is not fully complementary to a non-targetpolynucleotide in the sample, the method comprising the steps of: (a)contacting the sample with the primer combination and a polymerase underconditions that allow extension of a sequence from Pa which iscomplementary to the target polynucleotide when the targetpolynucleotide is present in the sample, (b) denaturing the sequenceextended from Pa from the target polynucleotide, and (c) repeating step(a) in the presence of a reverse primer having a sequence complementaryto a region in the sequence extended from Pa in step (b) to amplify thetarget polynucleotide, wherein extension and amplification of the targetpolynucleotide occurs when T₁ is fully complementary to the sequence inPa but is less efficient or does not occur when the first region in thetarget polynucleotide is not fully complementary to the sequence in Pa.In some aspects of the methods, the reverse primer has a sequence thatis fully complementary to a region in the sequence extended from Pa. Inother aspects, the reverse primer is a primer combination comprising afirst polynucleotide and a second polynucleotide, the firstpolynucleotide (PP) comprising a first domain (PPa) having a sequencethat is fully complementary to a first region (TT₁) in the sequenceextended from Pa in step (a) and a second domain (PPc) comprising aunique polynucleotide sequence, and the second polynucleotide (FF)comprising a first domain (FFb) that is complementary to a second region(TT₂) in the sequence extended from Pa in step (a) and a second domain(FFd) comprising a polynucleotide sequence sufficiently complementary toPPc such that PPc and FFd will hybridize under appropriate conditions.In certain aspects, the methods further comprise the step of detecting aproduct amplified in the method, and in other aspects, detection iscarried out using polymerase chain reaction, and/or detection is carriedout in real time.

In each method of the disclosure, an aspect is provided wherein thereverse primer is a primer combination as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structural relationship of the basic polynucleotidecombination (i.e., first polynucleotide and second polynucleotide)disclosed herein.

FIG. 2 depicts a primer combination comprising two three-way junctionswith three target binding domains a, g, and b.

FIG. 3 depicts polynucleotide combinations with stable four-way (A) andfive-way (B) junctions with two target binding domains.

FIG. 4 depicts a primer combination with a blocker polynucleotide. Asdepicted in FIG. 4A, the 5′ base of the blocker polynucleotide overlapswith, and is different than, the 3′ base of the first polynucleotide.The 5′ base of the blocker polynucleotide is not complementary to thetarget polynucleotide and is displaced upon extension of the firstpolynucleotide by a polymerase. As depicted in FIG. 4B, the 5′ base ofthe blocker polynucleotide overlaps with, and is different than, the 3′base of the first polynucleotide. The 5′ base of the blockerpolynucleotide is 100% complementary to the non-target polynucleotidewhile the 3′ base of the first polynucleotide is not complementary tothe non-target polynucleotide. In this configuration, the blockerpolynucleotide blocks extension of the first polynucleotide by apolymerase.

FIG. 5 depicts a primer combination with a probe polynucleotide. Asdepicted in FIG. 5A, the probe polynucleotide comprises a label at its5′ end and a quencher at its 3′ end.

FIG. 5B depicts the structural relationship of a probe polynucleotide incombination with a first/second polynucleotide pair and a blockerpolynucleotide.

FIG. 6 depicts the structural relationship of the basic polynucleotidecombination with a universal quencher polynucleotide. As depicted, theuniversal quencher polynucleotide is complementary to the second domainof the second polynucleotide and comprises a quencher at its 3′ endwhile the first polynucleotide comprises a label at its 5′ end.

FIGS. 7A-7F are schematics illustrating the polynucleotide combinationsas used in the polymerase chain reaction (PCR).

FIGS. 8A and 8B depict the structural relationship of the basicpolynucleotide combination and a reverse primer. In FIG. 8A, the reverseprimer is a single polynucleotide. In FIG. 8B, the reverse primer is asecond set of first and second polynucleotides.

FIG. 9A depicts a fluorophore-quencher labeled first polynucleotide(Primer A) with a non-specific RNA linker and a typical secondpolynucleotide (Fixer A). FIG. 9B illustrates the use of the combinationdepicted in FIG. 9A in PCR. When the strand opposite Primer A isgenerated, cleavage of the RNA-DNA hybrid by RNase H releases thefluorophore (or quencher) and a fluorescent signal is detected. FIG. 9Cdepicts a fluorophore-quencher labeled first polynucleotide (Primer A)with a site-specific RNA-DNA linker and a typical second polynucleotide(Fixer A). FIG. 9D illustrates the use of the combination depicted inFIG. 9C in PCR. When the PCR product comprising Primer A is denatured,the RNA-DNA linker hybridizes to a region downstream of Primer A, RNaseH cleaves the RNA-DNA hybrid and releases the fluorophore and asequence-specific fluorescent signal is detected.

FIG. 10 depicts primer combinations with three-way or four-way junctionsfor use in real-time PCR. With three-way junction primers, the primerpolynucleotide (i.e., first polynucleotide) is labeled with afluorophore on its 5′ end, and the fixer polynucleotide (i.e., secondpolynucleotide) is labeled with a quencher on its 3′ end. With four-wayjunction primers, the primer polynucleotide is labeled with afluorophore on its 5′ end, and the staple is labeled with a quencher onits 3′ end. The fixer polynucleotide is unlabeled. Since the seconddomain regions of both the primer and fixer polynucleotides are unique,the staple polynucleotide can be used as a “universal” quencherpolynucleotide.

FIG. 11 depicts three examples utilizing basic primer and fixerpolynucleotides with a non-covalently attached anti-primer (AP).

FIG. 12 depicts polynucleotides constructed with a basic primerpolynucleotide (“P0”) and a modified fixer polynucleotide structure (“F1through F4”). The modified polynucleotide combinations comprising thestem loop structures (1 through 4) may be utilized to provide anincreased level of specificity when binding to a templatepolynucleotide.

FIGS. 13A-13C depict polynucleotide combinations constructed with amodified primer polynucleotide (“P1”) and a basic fixer polynucleotide(“F”) FIG. 13A. In FIG. 13A, the modified primer polynucleotide is shownon the left, with the complete polynucleotide combination (modifiedprimer polynucleotide and basic fixer polynucleotide) shown on theright.

FIG. 14 depicts two scenarios for using the basic polynucleotidecombination (i.e., first polynucleotide and second polynucleotide)disclosed herein to detect point mutations. In the top scenario, thefirst domain of a primer polynucleotide is 100% complementary to thetemplate DNA polynucleotide and extension occurs, yielding a product. Inthe bottom scenario, the first domain of the primer polynucleotidecontains a mismatch relative to the template DNA polynucleotide andextension is blocked due to the instability in the short first domain ofthe primer polynucleotide. This instability will result in a very lowefficiency of PCR and will yield very little or no detectable product.

FIG. 15 depicts the use of a polynucleotide combination (i.e., firstpolynucleotide and second polynucleotide) to perform next generationsequencing (NGS).

FIG. 16 illustrates the results of quantitative PCR in the presence ofstaining dye SYTO 9 using the basic polynucleotide combination (i.e.,first polynucleotide and second polynucleotide) disclosed herein.

FIG. 17 illustrates the results of quantitative PCR assay withfluorophor-labeled Probe Primer (i.e., first polynucleotide) andquencher-labeled Fixer (i.e., second polynucleotide).

FIG. 18 illustrates the results of a qPCR assay using afluorophor-labeled Probe Primer (i.e., first polynucleotide) and auniversal quencher.

FIGS. 19A and 19B illustrate the results of a qPCR assay to detectmutant KRAS G12V in a mixture using a first polynucleotide, a Fixer(i.e., second polynucleotide) and staining with SYBR green dye.

FIG. 20 illustrates the results of a qPCR assay to detect mutant KRASG12V in formaldehyde-fixed samples using a first polynucleotide, a Fixer(i.e., second polynucleotide) and staining with SYBR green dye.

FIG. 21 illustrates the results of a qPCR assay to detect mutant KRASG12V in a mixture using a first polynucleotide, a Fixer (i.e., secondpolynucleotide) and a Probe Polynucleotide (i.e., TaqMan).

FIG. 22 illustrates the results of a qPCR assay to detect mutant KRASG12V in a mixture using a first polynucleotide, a Fixer (i.e., secondpolynucleotide), a Probe Polynucleotide (i.e., TaqMan), and a blockerpolynucleotide.

FIG. 23 illustrates the results of a qPCR assay to detect mutant KRASG12V in a mixture using a first polynucleotide modified with LNA at its3′ end, a Fixer (i.e., second polynucleotide), a Probe Polynucleotide(i.e., TaqMan), and a blocker polynucleotide.

FIGS. 24A-24D illustrate the results of a qPCR assay to detect mutantKRAS G12V in a mixture with 0.5 copy/reaction of KRAS G12V DNA(determined statistically) using a first polynucleotide modified withLNA at its 3′ end, a Fixer (i.e., second polynucleotide), a ProbePolynucleotide (i.e., TaqMan), and a blocker polynucleotide. FIGS.24E-24H illustrate the results of a qPCR assay to detect mutant KRASG12V in a mixture with no copies of KRAS G12V DNA using a firstpolynucleotide modified with LNA at its 3′ end, a Fixer (i.e., secondpolynucleotide), a Probe Polynucleotide (i.e., TaqMan), and a blockerpolynucleotide. 94% of WT DNA samples (15 out of 16) showed no signal,indicating a very high selectivity of single mutant DNA detection by theimproved KRAS G12V qPCR mutation assay.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery of a discontinuouspolynucleotide design that overcomes problems encountered during thehybridization of polynucleotides, and in particular, amplificationprimer hybridization to a target polynucleotide. These problems includebut are not limited to surmounting difficult secondary structure in thetarget polynucleotide and a low specificity to single-base changes in atarget polynucleotide.

Polynucleotide combinations described herein offer an advantage overboth standard PCR primers and long PCR primers when using polynucleotidetemplates that are difficult to amplify efficiently. Such templatesinclude, for example, those that contain a degree of secondary structureformed through internal self-hybridization giving rise to, for example,loops, hairpins and the like, that preclude, cause to be less efficientor inhibit hybridization to a complementary sequence. Template secondarystructure can prevent priming with a standard PCR primer which is unableto destabilize the internal hybridization and thus is unable tohybridize to the primer complement. Using polynucleotide combinations ofthe invention, template secondary structure is dehybridized (or melted)and hybridization with the complementary template regions occurs underappropriate conditions. As used herein, a “standard PCR primer” lengthcan be about 10 to about 100 bases.

A long PCR primer is able to resolve secondary structure in a targetpolynucleotide, but is not able to simultaneously provide either thespecificity or sensitivity near the 3′ (priming) end of the primer. Thisis because for a long PCR primer a large portion is hybridized to thetarget polynucleotide and a mismatch near the 3′ end of the primerrelative to the target polynucleotide will not be sufficient to reducepriming efficiency. As a result, a PCR product will still be synthesizeddespite the mismatch(s).

The polynucleotide combinations of the invention offer other advantages.For example, short PCR primers alone are useful for precise sequencehybridization to the target polynucleotide, but in order to achieve thehigh specificity of primer binding to a target polynucleotide that isdesired for PCR, the highest possible annealing temperature is typicallychosen. This annealing temperature is chosen based on the meltingtemperature of a given primer, and for a short primer that annealingtemperature will be relatively low. A low annealing temperature,however, has the disadvantage of allowing for non-specific hybridizationof the short primer to the target polynucleotide, resulting innon-specific PCR product formation. Based on the relatively lowannealing temperature that must be used to allow a short PCR primer toanneal to its target polynucleotide, short primers form duplexes with atarget polynucleotide that are typically unstable even when they are100% complementary to the target polynucleotide region. Moreover, theseduplexes are even more unstable when the primer is less than 100%complementary (i.e., at least one mismatch between the primer and thetarget polynucleotide region). The polynucleotide combination of theinvention helps to overcome the instability problem associated withusing a short PCR primer and permit highly specific binding to a desiredtarget. For example, combinations of the disclosure are able todiscriminate between target sequences that differ by as little as asingle base.

For example, the discontinuous polynucleotide combination design (seeFIG. 1) allows for use of a short PCR primer region [Pa] throughhybridization of the first domain [Fa] of the fixer polynucleotide(i.e., “second polynucleotide”) to the temple polynucleotide andhybridization of the second domain [Pc] of the primer polynucleotide(i.e., “first polynucleotide”) to the second domain [Fd] of the fixerpolynucleotide, thereby giving the effective result of an apparent“longer” primer sequence. This longer and discontinuous hybridization ineffect stabilizes binding between the first region [Pa] of the primerpolynucleotide even if this region is as small as eight bases, therebyincreasing the efficiency of PCR.

In another embodiment, the regions of the template polynucleotide thatare complementary to the first domain of the primer polynucleotide andfixer polynucleotide need not be directly adjacent. The presentinvention contemplates embodiments wherein the complementary regions areseparated (i.e., discontinuous) by up to 10 nucleotides or more, andthat upon fixer hybridization to the template polynucleotide, theintervening sequence is looped-out to bring the target template regionto be amplified into proximity with the primer polynucleotide. In thisaspect then, the primer combination actually induces secondary structurein the template polynucleotide, with or without internalself-hybridization of the looped out structure.

I. Polynucleotide Primer Combinations

In an embodiment, a polynucleotide combination is provided comprising afirst polynucleotide and a second polynucleotide, the firstpolynucleotide comprising a first domain [Pa] that is complementary to afirst target polynucleotide region and a second domain [Pc] comprising aunique polynucleotide sequence, and the second polynucleotide comprisinga first domain [Fb] that is complementary to a second targetpolynucleotide region and a second domain [Fd] comprising apolynucleotide sequence sufficiently complementary to the second domainof the first polynucleotide such that the second domain of the firstpolynucleotide and the second domain of the second polynucleotide willhybridize under appropriate conditions. The structural relationship ofthe basic polynucleotide combination is shown in FIG. 1.

A. Three-Way Junction

In a three-way junction polynucleotide combination, the primerpolynucleotide and the fixer polynucleotide are associated throughinteraction between the second domain of the first polynucleotide (Pc inScheme 1) and the second domain of the second polynucleotide (Fd inScheme 1), and the first domain of the primer oligonucleotide (Pa inFIG. 1) and the first domain of the fixer polynucleotide (Fb in FIG. 1)are hybridized to respective complementary regions in the targetpolynucleotide as shown in FIG. 2 (Scheme 2A).

B. More Than Three-Way Junctions

The invention also contemplates an alternative embodiment wherein theprimer combination comprises two three-way junctions (see FIG. 2; Scheme2B). In some of these embodiments, domain “e” is complementary to domain[Pc], domain “f” is complementary to domain [Fd], and domain “g” iscomplementary to a third target polynucleotide region.

The present invention further contemplates a polynucleotide combinationthat comprises a four-way junction. In a four-way junctionpolynucleotide combination the primer polynucleotide and fixerpolynucleotide are associated through a “staple” polynucleotide (Scheme3A, below). The staple polynucleotide is able to hybridize with thesecond domains of both the primer polynucleotide and the fixerpolynucleotide. The structure of the staple polynucleotide comprises afirst domain [e] that is sufficiently complementary to the second domain[Pc] of the primer polynucleotide so that it the regions can hybridizeunder appropriate conditions, and a second domain [f] that issufficiently complementary to the second domain [Fd] of the fixerpolynucleotide can hybridize such that the regions can hybridize underappropriate conditions. In some aspects of this embodiment, the seconddomain Pc of the primer polynucleotide need not be sufficientlycomplementary to the second domain Fd in the fixer polynucleotide so asto allow for hybridization between the Pc domain and the Fd domain undertypical conditions. In this aspect, with the first domain [e] in thestaple being sufficiently complementary to the second domain Pc of theprimer polynucleotide, and the second domain [f] of the staple beingsufficiently complementary to the second domain [Fd] of the fixerdomain, allows for formation of the stable four way junction shown inFIG. 3 (Scheme 3A).

Additional “staple” polynucleotides in the polynucleotide combinationsare also contemplated as described herein, thereby generatingmultiple-way junctions depending on the number of staple polynucleotidesin the polynucleotide combination. Scheme 3B shows a five way junctionand the person of skill in the art will readily appreciate how junctionslarger than five will be constructed.

Further, the “staple” polynucleotide may comprise a modified nucleotideas described herein. In additional aspects, the “staple” polynucleotidemay comprise a label and/or a quencher. In these aspects, the label orquencher may be on either the 5′ or 3′ end of the “staple”polynucleotide.

C. Primer Combinations Comprising a Blocker Polynucleotide

The invention also contemplates embodiments wherein a blockerpolynucleotide is included with a polynucleotide combinations. A blockerpolynucleotide has a sequence that is complementary to a targetpolynucleotide region located immediately 5′ of the first targetpolynucleotide region. This is depicted in FIG. 4. In some embodiments,the blocker polynucleotide overlaps with the first domain of the firstpolynucleotide. In other words, the nucleotide(s) at the 3′ end of thefirst polynucleotide and the nucleotide(s) at the 5′ end of the blockerpolynucleotide would be complementary to the same nucleotide(s) of thetarget polynucleotide. In various embodiments, the overlap of the firstpolynucleotide and the blocker polynucleotide is 1 nucleotide, 2nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides,7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15nucleotides. In related embodiments, the nucleotide(s) at the 3′ end ofthe first polynucleotide and the nucleotide(s) at the 5′ end of theblocker polynucleotide are different. In these embodiments, thenucleotide(s) at the 3′ end of the first polynucleotide would hybridizeto the target polynucleotide when they are complementary to the targetpolynucleotides at the appropriate position, thus allowing for extensionof the first polynucleotide under the appropriate conditions (see FIG.4a ). In related embodiments, the nucleotide(s) at the 5′ end of theblocker polynucleotide would hybridize to the target polynucleotide whenthey are complementary to the non-target polynucleotide at theappropriate position, thus blocking extension of the firstpolynucleotide (see FIG. 4b ). In various embodiments, the nucleotide atthe 3′ end of the blocker polynucleotide is modified to preventextension by a polymerase.

In some embodiments, the overlapping sequences of the blockerpolynucleotide and the first domain of the first polynucleotide (Pa)differ by at least 2 bases, at least 3 bases, at least 4 bases, at least5 bases, at least 6 bases, at least 7 bases, at least 8 bases, at 9 twobases, or by at least 10 bases. The differing bases can be at anyposition in the overlapping portions.

D. Primer Combinations Comprising a Probe Polynucleotide

The invention also contemplates embodiments wherein a probepolynucleotide is included with the above polynucleotide combinations. Aprobe polynucleotide has a sequence that is complementary to a targetpolynucleotide region located 5′ of the first target polynucleotideregion (see FIG. 5a ). In other embodiments, a probe polynucleotide hasa sequence that is complementary to the extension product of the firstpolynucleotide (see FIG. 5b ). As is apparent, this probe polynucleotidewould be complementary to the complementary strand of the targetpolynucleotide. In embodiments wherein a blocker polynucleotide isincluded in the primer combination with the probe polynucleotide, theprobe polynucleotide is complementary to a target polynucleotide regionlocated 5′ of the target polynucleotide region complementary to theblocker polynucleotide. In various embodiments, the probe polynucleotidecomprises a label at its 5′ end. In related embodiments, the probepolynucleotide further comprises a quencher at its 3′ end. In stillfurther embodiments, the probe polynucleotide further comprises aninternal quencher, such as, and without limitation, the Zen quencher.

E. Primer Combinations Comprising a Universal Quencher

In various embodiments, the primer polynucleotide combination includes auniversal quencher polynucleotide. The universal quencher polynucleotideis complementary to the second domain of the second polynucleotide suchthat it hybridizes to the second domain of the second polynucleotide. Inthese embodiments, the universal quencher polynucleotide hybridizes to aregion of the second polynucleotide located 3′ of the region to whichthe second domain of the second polynucleotide hybridizes (see FIG. 6).The universal quencher polynucleotide is labeled at its 3′ end with aquencher.

F. Primer Combinations Comprising a Reverse Primer

The invention also contemplates embodiments wherein a reverse primerpolynucleotide is included with the above polynucleotide combinations.The reverse primer is complementary to a region in the polynucleotidecreated by extension of the first polynucleotide (see FIG. 8a ). As isapparent, in some embodiments the reverse primer is also complementaryto the complementary strand of the target polynucleotide when the targetpolynucleotide is one strand of a double-stranded polynucleotide. Insome embodiments, the reverse primer is a combination firstpolynucleotide/second polynucleotide, as described above (see FIG. 8b ).

II. Polynucleotides

As used herein, the term “polynucleotide,” either as a component of apolynucleotide pair combination, including blocker polynucleotides andprobes, or as a target molecule, is used interchangeably with the termoligonucleotide.

The term “nucleotide” or its plural as used herein is interchangeablewith modified forms as discussed herein and otherwise known in the art.In certain instances, the art uses the term “nucleobase” which embracesnaturally-occurring nucleotides as well as modifications of nucleotidesthat can be polymerized.

Methods of making polynucleotides of a predetermined sequence arewell-known in the art. See, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotidesand Analogues, 1st Ed. (Oxford University Press, New York, 1991).Solid-phase synthesis methods are preferred for botholigoribonucleotides and oligodeoxyribonucleotides (the well-knownmethods of synthesizing DNA are also useful for synthesizing RNA).Oligoribonucleotides and oligodeoxyribonucleotides can also be preparedenzymatically.

In various aspects, methods provided include use of polynucleotideswhich are DNA oligonucleotides, RNA oligonucleotides, or combinations ofthe two types. Modified forms of oligonucleotides are also contemplatedwhich include those having at least one modified internucleotidelinkage. Modified polynucleotides or oligonucleotides are described indetail herein below.

III. Modified Polynucleotide

Specific examples of oligonucleotides include those containing modifiedbackbones or non-natural internucleoside linkages. Oligonucleotideshaving modified backbones include those that retain a phosphorus atom inthe backbone and those that do not have a phosphorus atom in thebackbone. Modified oligonucleotides that do not have a phosphorus atomin their internucleoside backbone are considered to be within themeaning of “oligonucleotide.” In specific embodiments, the firstpolynucleotide comprises phosphorothioate linkages.

Modified oligonucleotide backbones containing a phosphorus atom include,for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Also contemplated are oligonucleotides having inverted polaritycomprising a single 3′ to 3′ linkage at the 3′-most internucleotidelinkage, i.e. a single inverted nucleoside residue which may be abasic(the nucleotide is missing or has a hydroxyl group in place thereof).Salts, mixed salts and free acid forms are also contemplated.Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, U.S. Pat. Nos. 3,687,808;4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599;5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, thedisclosures of which are incorporated by reference herein.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages; siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; riboacetyl backbones;alkene containing backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts. See,for example, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134;5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704;5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and5,677,439, the disclosures of which are incorporated herein by referencein their entireties.

In still other embodiments, oligonucleotide mimetics wherein both one ormore sugar and/or one or more internucleotide linkage of the nucleotideunits are replaced with “non-naturally occurring” groups. In one aspect,this embodiment contemplates a peptide nucleic acid (PNA). In PNAcompounds, the sugar-backbone of an oligonucleotide is replaced with anamide containing backbone. See, for example U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, and Nielsen et al., 1991, Science, 254:1497-1500, the disclosures of which are herein incorporated byreference.

In still other embodiments, oligonucleotides are provided withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and including —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂—,—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—described in U.S. Pat. Nos. 5,489,677, and 5,602,240. Also contemplatedare oligonucleotides with morpholino backbone structures described inU.S. Pat. No. 5,034,506.

In various forms, the linkage between two successive monomers in theoligo consists of 2 to 4, desirably 3, groups/atoms selected from —CH₂—,—O—, —S—, —NR^(H)—, >C═O, >C═NR^(H), >C═S, —Si(R″)₂—, —SO—, —S(O)₂—,—P(O)₂—, —PO(BH₃)—, —P(O,S)—, P(S)₂—, —PO(R″)—, —PO(OCH₃)—, and—PO(NHR^(H))—, where RH is selected from hydrogen and C₁₋₄-alkyl, and R″is selected from C₁₋₆-alkyl and phenyl. Illustrative examples of suchlinkages are —CH₂—CH₂—CH₂—, —CH₂—CO—CH₂—, —CH₂—CHOH—CH₂—, O—CH₂—O—,—O—CH₂—CH₂—, —O—CH₂—CH=(including R⁵ when used as a linkage to asucceeding monomer), —CH₂—CH₂—O—, —NR^(H)—CH₂—CH₂—, —CH₂CH₂—NR^(H),—CH₂—NR^(H)—CH₂—, —O—CH₂—CH₂—NR^(H)—, —NR^(H)—CO—O—, —NR^(H)—CO—NR^(H)—,—NR^(H)—CS—NR^(H)—, —NR^(H)—C(═NR^(H))—NR^(H)—,—NR^(H)—CO—CH₂—NR^(H)—O—CO—O—, —O—CO—CH₂—O—, —O—CH₂—CO—O—,—CH₂—CO—NR^(H)—, —O—CO—NR^(H), —NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H)—,—O—CH₂—CH₂—NR^(H)—, —CH═N—O—, —CH₂—NR^(H)—O—, —CH₂—O—N=(including R⁵when used as a linkage to a succeeding monomer), —CH₂—O—NR^(H)—,—CO—NR^(H)—CH₂—, —CH₂—NR^(H)—O—, CH₂—NR^(H)—CO—, —O—NR^(H)—CH₂—,—O—NR^(H), —O—CH₂—S—, —S—CH₂—O—, —CH₂— CH₂—S—, —O— CH₂— CH₂—S—, —S—CH₂—CH=(including R⁵ when used as a linkage to a succeeding monomer),—S— CH₂— CH₂—, —S— CH₂— CH₂—O—, —S—CH₂— CH₂—S—, —CH₂—S— CH₂—, —CH₂—SO—CH₂—, —CH₂—SO₂— CH₂—, O—SO—O—, —O—S(O)₂—O—, —O—S(O)₂— CH₂—,—O—S(O)₂—NR^(H)—, —NR^(H)—S(O)₂— CH₂—; —O—S(O)₂— CH₂—, —O—P(O)₂—O—,—O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—,—O—P(O)₂—S—, —O—P(O,S)—S—, —O—P(S)₂—S—, —S—P(O)₂—S—, —S—P(O,S)—S—,—S—P(S)₂—S—, —O—PO(R″)—O—, —O—PO(OCH₃)—O—, —O—PO(O CH₂CH₃)—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—, —O—PO(NHR^(N))—O—, —O—P(O)₂—NR^(H) H—,—NR^(H)—P(O)₂—O—_, —O—P(O,NR^(H))—O—, —CH₂—P(O)₂—O—, —O—P(O)₂—CH₂—, and—O—Si(R″)₂—O—; among which —CH₂—CO—NR^(H)—, —CH₂—NR^(H)—O—, —S— CH₂—O—,—O—P(O)₂—O—O—P(—O,S)—O—, —O—P(S)₂—O—, —NR^(H) P(O)₂—O—,—O—P(O,NR^(H))—O—, —O—PO(R″)—O—, —O—PO(CH₃)—O—, and —O—PO(NHR^(N))—O—,where RH is selected form hydrogen and C₁₋₄-alkyl, and R″ is selectedfrom C₁₋₆-alkyl and phenyl, are contemplated. Further illustrativeexamples are given in Mesmaeker et. al., 1995, Current Opinion inStructural Biology, 5: 343-355 and Susan M. Freier and Karl-HeinzAltmann, 1997, Nucleic Acids Research, vol 25: pp 4429-4443.

Still other modified forms of oligonucleotides are described in detailin U.S. patent application NO. 20040219565, the disclosure of which isincorporated by reference herein in its entirety.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. In certain aspects, oligonucleotides comprise one of thefollowing at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Other embodiments includeO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other oligonucleotides comprise one of the following atthe 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃,OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.In one aspect, a modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., 1995, Helv. Chin. Acta, 78: 486-504) i.e., an alkoxyalkoxygroup. Other modifications include 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examplesherein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples herein below.

Still other modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂),2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modificationmay be in the arabino (up) position or ribo (down) position. In oneaspect, a 2′-arabino modification is 2′-F. Similar modifications mayalso be made at other positions on the oligonucleotide, for example, atthe 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′linked oligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. See, for example, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; 5,792,747; and 5,700,920, the disclosures of whichare incorporated by reference in their entireties herein.

In various aspects, a modification of the sugar includes Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.The linkage in certain aspects is a methylene (—CH₂—)_(n) group bridgingthe 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226, thedisclosures of which are incorporated by reference in their entiretiesherein. In various embodiments, the first polynucleotide comprises alocked nucleic acid. In some embodiments, the first polynucleotidecomprises a plurality of locked nucleic acids. In specific embodiments,the first domain of the first polynucleotide comprises a plurality oflocked nucleic acids. In more specific embodiments, the nucleotide atthe 3′ end of the first polynucleotide comprises a locked nucleic acid.In various embodiments, the blocker polynucleotide comprises a lockednucleic acid. In other embodiments, the blocker polynucleotide comprisesa plurality of locked nucleic acids. In specific embodiments, thenucleotide at the 5′ end of the blocker polynucleotide comprises alocked nucleic acid.

Polynucleotides may also include base modifications or substitutions. Asused herein, “unmodified” or “natural” bases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified bases include other synthetic andnatural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and3-deazaguanine and 3-deazaadenine. Further modified bases includetricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzox-azin-2(3H)-one),carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiedbases may also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further bases includethose disclosed in U.S. Pat. No. 3,687,808, those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed byEnglisch et al., 1991, Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these bases are useful for increasingthe binding affinity and include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. and are, in certain aspects combinedwith 2′-O-methoxyethyl sugar modifications. See, U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985;5,830,653; 5,763,588; 6,005,096; 5,750,692 and 5,681,941, thedisclosures of which are incorporated herein by reference.

A “modified base” or other similar term refers to a composition whichcan pair with a natural base (e.g., adenine, guanine, cytosine, uracil,and/or thymine) and/or can pair with a non-naturally occurring base. Incertain aspects, the modified base provides a T_(m) differential of 15,12, 10, 8, 6, 4, or 2° C. or less. Exemplary modified bases aredescribed in EP 1 072 679 and WO 97/12896.

By “nucleobase” is meant the naturally occurring nucleobases adenine(A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well asnon-naturally occurring nucleobases such as xanthine, diaminopurine,8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine,N⁴,N⁴-ethanocytosin, N′,N′-ethano-2,6-diaminopurine, 5-methylcytosine(mC), 5-(C³-C⁶)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-tr-iazolopyridin, isocytosine,isoguanine, inosine and the “non-naturally occurring” nucleobasesdescribed in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freierand Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp4429-4443. The term “nucleobase” thus includes not only the known purineand pyrimidine heterocycles, but also heterocyclic analogues andtautomers thereof. Further naturally and non-naturally occurringnucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan,et al.), in Chapter 15 by Sanghvi, in Antisense Research andApplication, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, inEnglisch et al., 1991, Angewandte Chemie, International Edition, 30:613-722 (see especially pages 622 and 623, and in the ConciseEncyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed.,John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design1991, 6, 585-607, each of which are hereby incorporated by reference intheir entirety). The term “nucleosidic base” or “base unit” is furtherintended to include compounds such as heterocyclic compounds that canserve like nucleobases including certain “universal bases” that are notnucleosidic bases in the most classical sense but serve as nucleosidicbases. Especially mentioned as universal bases are 3-nitropyrrole,optionally substituted indoles (e.g., 5-nitroindole), and optionallysubstituted hypoxanthine. Other desirable universal bases include,pyrrole, diazole or triazole derivatives, including those universalbases known in the art.

IV. Polynucleotide Structure—Length

In one aspect, the first domain of the first polynucleotide is 5nucleotides that is complementary to a target polynucleotide region. Invarious aspects, the first domain of the first polynucleotide is atleast 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, atleast 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides,at least 12 nucleotides, at least 13 nucleotides, at least 14nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, atleast 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides,at least 23 nucleotides, at least 24 nucleotides, at least 25nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least28 nucleotides, at least 29 nucleotides, at least 30 nucleotides or morethat is complementary to a target polynucleotide region. In a relatedaspect, the second domain of the first polynucleotide comprises 10 ormore nucleotides in a unique DNA sequence that is sufficientlycomplementary to the second domain of the second polynucleotide so as toallow hybridization between these two complementary sequences underappropriate conditions. In various aspects, the second domain of thefirst polynucleotide comprises at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24nucleotides, at least 25, at least about 30, at least about 35, at leastabout 40, at least about 45, at least about 50, at least about 60, atleast about 70, at least about 80, at least about 90, at least about100, at least about 120, at least about 140, at least about 160, atleast about 180, at least about 200, at least about 220, at least about240, at least about 260, at least about 280, at least about 300, atleast about 320, at least about 340, at least about 360, at least about380, at least about 400, at least about 420, at least about 440, atleast about 460, at least about 480, at least about 500 or morenucleotides of a unique DNA sequence that is sufficiently complementaryto the second domain of the second polynucleotide so as to allowhybridization between the two complementary sequences under appropriateconditions.

In another embodiment, the second polynucleotide comprises a firstdomain containing about 10 nucleotides, this first domain of the secondpolynucleotide being complementary to a target DNA region that isdifferent from the target region recognized by the first domain of thefirst polynucleotide. In various aspects, the second polynucleotidecomprises a first domain containing at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, 19,at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25, at least 26, at least 27, at least 28, at least 29, at least30, at least 31, at least 32, at least 33, at least 34, at least 35, atleast 36, at least 37, at least 38, at least 39, at least 40, at least41, at least 42, at least 43, at least 44, at least 45, at least 46, atleast 47, at least 48, at least 49, at least 50, at least about 100, atleast about 150, at least about 200, at least about 250, at least about300, at least about 350, at least about 400, at least about 450, atleast about 500, at least about 550, at least about 600, at least about650, at least about 700, at least about 750, at least about 800, atleast about 850, at least about 900, at least about 950, at least about1000, at least about 1100, at least about 1200, at least about 1300, atleast about 1400, at least about 1500, at least about 1600, at leastabout 1700, at least about 1800, at least about 1900, at least about2000, at least about 2100, at least about 2200, at least about 2300, atleast about 2400, at least about 2500, at least about 2600, at leastabout 2700, at least about 2800, at least about 2900, at least about3000, at least about 3100, at least about 3200, at least about 3300, atleast about 3400, at least about 3500, at least about 3600, at leastabout 3700, at least about 3800, at least about 3900, at least about4000, at least about 4100, at least about 4200, at least about 4300, atleast about 4400, at least about 4500, at least about 4600, at leastabout 4700, at least about 4800, at least about 4900, at least about5000 or more nucleotides, the first domain of this second polynucleotidebeing complementary, or sufficiently complementary, so as to recognizeand bind to a target DNA region that is different from the target regionrecognized by the first domain of the first polynucleotide.

In a related aspect, the second domain of the second polynucleotidecomprises 10 nucleotides of a unique DNA sequence that is sufficientlycomplementary to the second domain of the first polynucleotide so as toallow hybridization under appropriate conditions. In various aspects,the second domain of the second polynucleotide comprises at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least about 30, at least about35, at least 40, at least about 45, at least about 50, at least about60, at least about 70, at least about 80, at least about 90, at leastabout 100, at least about 120, at least about 140, at least about 160,at least about 180, at least about 200, at least about 220, at leastabout 240, at least about 260, at least about 280, at least about 300,at least about 320, at least about 340, at least about 360, at leastabout 380, at least about 400, or least about 420, or least about 440,at least about 460, at least about 480, at least about 500 or morenucleotides of a unique DNA sequence that is sufficiently complementaryto the second domain of the first polynucleotide so as to allowhybridization between the two sufficiently complementary sequences underappropriate conditions.

In some embodiments, compositions and methods described herein include asecond set of polynucleotides with the characteristics described abovefor first and second polynucleotides. In some embodiments, a pluralityof sets is contemplated. These additional sets of first and secondpolynucleotides can have any of the characteristics described for firstand second polynucleotides.

The “staple” polynucleotides, as depicted in FIG. 3 (Scheme 3A and 3B),are contemplated in one aspect to comprise at least 20 nucleotides. Inother aspects, the “staple” polynucleotides can comprise at least 21nucleotides, or at least 22 nucleotides, or at least 23 nucleotides, orat least 24 nucleotides, or at least 25 nucleotides, or at least 26nucleotides, or at least 27 nucleotides, or at least 28 nucleotides, orat least 29 nucleotides, or at least 30 nucleotides, or at least about35 nucleotides, or at least about 40 nucleotides, or at least about 45nucleotides, or at least about 50 nucleotides, or at least about 55nucleotides, or at least about 60 nucleotides, or at least about 65nucleotides, or at least about 70 nucleotides, or at least about 75nucleotides, or at least about 80 nucleotides, or at least about 85nucleotides, or at least about 90 nucleotides, or at least about 95nucleotides, or at least about 100 nucleotides, or at least about 150nucleotides, or at least about 200 nucleotides, or at least about 250nucleotides, or at least about 300 nucleotides, or at least about 350nucleotides, or at least about 400 nucleotides, or at least about 450nucleotides, or at least 500 nucleotides or more.

In some embodiments, the universal quencher polynucleotide is from about5 nucleotides in length to about 100 bases in length. In variousaspects, the universal quencher polynucleotide comprises at least 5nucleotides, or at least 6 nucleotides, or at least 7 nucleotides, or atleast 8 nucleotides, or at least 9 nucleotides, or at least 10nucleotides, or at least 11 nucleotides, or at least 12 nucleotides, orat least 13 nucleotides, or at least 14 nucleotides, or at least 15nucleotides, or at least 16 nucleotides, or at least 17 nucleotides, orat least 18 nucleotides, or at least 19 nucleotides, or at least 20nucleotides, or at least 21 nucleotides, or at least 22 nucleotides, orat least 23 nucleotides, or at least 24 nucleotides, or at least 25nucleotides, or at least 26 nucleotides, or at least 27 nucleotides, orat least 28 nucleotides, or at least 29 nucleotides, or at least 30nucleotides, or at least about 35 nucleotides, or at least about 40nucleotides, or at least about 45 nucleotides, or at least about 50nucleotides, or at least about 55 nucleotides, or at least about 60nucleotides, or at least about 65 nucleotides, or at least about 70nucleotides, or at least about 75 nucleotides, or at least about 80nucleotides, or at least about 85 nucleotides, or at least about 90nucleotides, or at least about 95 nucleotides, or at least about 100nucleotides of a unique DNA sequence that is sufficiently complementaryto the second domain of the second polynucleotide so as to allowhybridization under appropriate conditions.

In some embodiments, the probe polynucleotide is from about 5nucleotides in length to about 100 bases in length. In various aspects,the probe polynucleotide comprises at least 5 nucleotides, or at least 6nucleotides, or at least 7 nucleotides, or at least 8 nucleotides, or atleast 9 nucleotides, or at least 10 nucleotides, or at least 11nucleotides, or at least 12 nucleotides, or at least 13 nucleotides, orat least 14 nucleotides, or at least 15 nucleotides, or at least 16nucleotides, or at least 17 nucleotides, or at least 18 nucleotides, orat least 19 nucleotides, or at least 20 nucleotides, or at least 21nucleotides, or at least 22 nucleotides, or at least 23 nucleotides, orat least 24 nucleotides, or at least 25 nucleotides, or at least 26nucleotides, or at least 27 nucleotides, or at least 28 nucleotides, orat least 29 nucleotides, or at least 30 nucleotides, or at least 31nucleotides, or at least 32 nucleotides, or at least 33 nucleotides, orat least 34 nucleotides, or at least 35 nucleotides, or at least 36nucleotides, or at least 37 nucleotides, or at least 38 nucleotides, orat least 39 nucleotides, or at least 40 nucleotides, or at least about45 nucleotides, or at least about 50 nucleotides, or at least about 55nucleotides, or at least about 60 nucleotides, or at least about 65nucleotides, or at least about 70 nucleotides, or at least about 75nucleotides, or at least about 80 nucleotides, or at least about 85nucleotides, or at least about 90 nucleotides, or at least about 95nucleotides, or at least about 100 nucleotides of a DNA sequence that issufficiently complementary to a target polynucleotide region so as toallow hybridization under appropriate conditions.

In some embodiments, the blocker polynucleotide is from about 5nucleotides in length to about 100 bases in length. In various aspects,the blocker polynucleotide comprises at least 5 nucleotides, or at least6 nucleotides, or at least 7 nucleotides, or at least 8 nucleotides, orat least 9 nucleotides, or at least 10 nucleotides, or at least 11nucleotides, or at least 12 nucleotides, or at least 13 nucleotides, orat least 14 nucleotides, or at least 15 nucleotides, or at least 16nucleotides, or at least 17 nucleotides, or at least 18 nucleotides, orat least 19 nucleotides, or at least 20 nucleotides, or at least 21nucleotides, or at least 22 nucleotides, or at least 23 nucleotides, orat least 24 nucleotides, or at least 25 nucleotides, or at least 26nucleotides, or at least 27 nucleotides, or at least 28 nucleotides, orat least 29 nucleotides, or at least 30 nucleotides, or at least 31nucleotides, or at least 32 nucleotides, or at least 33 nucleotides, orat least 34 nucleotides, or at least 35 nucleotides, or at least 36nucleotides, or at least 37 nucleotides, or at least 38 nucleotides, orat least 39 nucleotides, or at least 40 nucleotides, or at least about45 nucleotides, or at least about 50 nucleotides, or at least about 55nucleotides, or at least about 60 nucleotides, or at least about 65nucleotides, or at least about 70 nucleotides, or at least about 75nucleotides, or at least about 80 nucleotides, or at least about 85nucleotides, or at least about 90 nucleotides, or at least about 95nucleotides, or at least about 100 nucleotides of a polynucleotidesequence that is sufficiently complementary to a target polynucleotideregion so as to allow hybridization under appropriate conditions. Invarious embodiments, the blocker polynucleotide further comprises amodified nucleic acid as the nucleotide at its 5′ end. In variousembodiments, the modified nucleic acid is a locked nucleic acid. In someembodiments, the blocker polynucleotide further comprises a blockinggroup at the 3′ end to prevent extension by a polymerase.

In some embodiments, the reverse primer polynucleotide is from about 5nucleotides in length to about 100 bases in length. In various aspects,the reverse primer polynucleotide comprises at least 5 nucleotides, orat least 6 nucleotides, or at least 7 nucleotides, or at least 8nucleotides, or at least 9 nucleotides, or at least 10 nucleotides, orat least 11 nucleotides, or at least 12 nucleotides, or at least 13nucleotides, or at least 14 nucleotides, or at least 15 nucleotides, orat least 16 nucleotides, or at least 17 nucleotides, or at least 18nucleotides, or at least 19 nucleotides, or at least 20 nucleotides, orat least 21 nucleotides, or at least 22 nucleotides, or at least 23nucleotides, or at least 24 nucleotides, or at least 25 nucleotides, orat least 26 nucleotides, or at least 27 nucleotides, or at least 28nucleotides, or at least 29 nucleotides, or at least 30 nucleotides, orat least 31 nucleotides, or at least 32 nucleotides, or at least 33nucleotides, or at least 34 nucleotides, or at least 35 nucleotides, orat least 36 nucleotides, or at least 37 nucleotides, or at least 38nucleotides, or at least 39 nucleotides, or at least 40 nucleotides, orat least about 45 nucleotides, or at least about 50 nucleotides, or atleast about 55 nucleotides, or at least about 60 nucleotides, or atleast about 65 nucleotides, or at least about 70 nucleotides, or atleast about 75 nucleotides, or at least about 80 nucleotides, or atleast about 85 nucleotides, or at least about 90 nucleotides, or atleast about 95 nucleotides, or at least about 100 nucleotides of apolynucleotide sequence that is sufficiently complementary to a regionof a polymerase-extended first polynucleotide so as to allowhybridization under appropriate conditions. In some embodiments, whenthe target polynucleotide is a double-stranded polynucleotide, thereverse primer is complementary to a complementary strand of the targetpolynucleotide. In some embodiments, the reverse primer is a combinationof first and second polynucleotides, as defined herein.

V. Polynucleotide Base Structure

In some embodiments, the first polynucleotide is comprised of DNA,modified DNA, RNA, modified RNA, PNA, or combinations thereof. In otherembodiments, the second polynucleotide is comprised of DNA, modifiedDNA, RNA, modified RNA, PNA, or combinations thereof.

VI. Polynucleotide Structure—Blocking Groups

Blocking groups are incorporated as needed when polymerase extensionfrom a 3′ region of a polynucleotide is undesirable. For example, thesecond domain of the second polynucleotide, in another aspect, furthercomprises a blocking group (“R” in FIG. 1) at the 3′ end of the seconddomain to prevent extension by an enzyme that is capable of synthesizinga nucleic acid. In additional aspects, the universal quencher comprisesa blocking group at its 3′ end. In further aspects, the blockerpolynucleotide comprises a blocking group at its 3′ end. Blocking groupsuseful in the practice of the methods include but are not limited to a3′ phosphate group, a 3′ amino group, a dideoxy nucleotide, a six carbonglycol spacer (and in one aspect the six carbon glycol spacer ishexanediol) and inverted deoxythymidine (dT).

VII. Polynucleotide Structure—Complementarity

In some aspects, the second domain of the second polynucleotide is atleast about 70% complementary to the second domain of the firstpolynucleotide. In related aspects, the second domain of the secondpolynucleotide is at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, or about 100% complementaryto the second domain of the first polynucleotide.

In one aspect, the second domain of the third polynucleotide is at leastabout 70% complementary to the second domain of the fourthpolynucleotide. In related aspects, the second domain of the thirdpolynucleotide is at least about 75%, or at least about 80%, or at leastabout 85%, or at least about 90%, or at least about 95%, or about 100%complementary to the second domain of the fourth polynucleotide.

In another aspect, the blocker polynucleotide is at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, or about 100% complementary to a sequence in the targetpolynucleotide, and in yet another aspect, the probe polynucleotide isat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, or about 100% complementary to a sequencein the target polynucleotide,

VIII. Hybridization Conditions

In some embodiments, the first and second polynucleotide hybridize toeach other under stringent conditions in the absence of a templatepolynucleotide. In some embodiments, the first and secondpolynucleotides do not hybridize to each other under stringentconditions in the absence of a template polynucleotide. “Stringentconditions” as used herein can be determined empirically by the workerof ordinary skill in the art and will vary based on, e.g., the length ofthe primer, complementarity of the primer, concentration of the primer,the salt concentration (i.e., ionic strength) in the hybridizationbuffer, the temperature at which the hybridization is carried out,length of time that hybridization is carried out, and presence offactors that affect surface charge of the polynucleotides. In general,stringent conditions are those in which the polynucleotide is able tobind to its complementary sequence preferentially and with higheraffinity relative to any other region on the target. Exemplary stringentconditions for hybridization to its complement of a polynucleotidesequence having 20 bases include without limitation about 50% G+Ccontent, 50 mM salt (Nat), and an annealing temperature of 60° C. For alonger sequence, specific hybridization is achieved at highertemperature. In general, stringent conditions are such that annealing iscarried out about 5° C. below the melting temperature of thepolynucleotide. The “melting temperature” is the temperature at which50% of polynucleotides that are complementary to a target polynucleotidein equilibrium at definite ion strength, pH and polynucleotideconcentration.

IX. Methods of Use A. PCR

In target polynucleotide amplification methods described herein, a thirdpolynucleotide and a fourth polynucleotide are contemplated for use incombination with the polynucleotide combination described above, thethird polynucleotide comprising a first domain [Pa] that iscomplementary to a complementary strand of the target polynucleotide[relative to the strand to which the first domain of the firstpolynucleotide is complementary] at a first target complementpolynucleotide region and a second domain [Pc] comprising a uniquepolynucleotide sequence, and the fourth polynucleotide comprising afirst domain [Fb] that is complementary to the complementary strand ofthe target polynucleotide [relative to the strand to which the secondpolynucleotide is complementary] at a second complement targetpolynucleotide region and a second domain [Fd] comprising apolynucleotide sequence sufficiently complementary to the second domainof the third polynucleotide such that the second domain of the thirdpolynucleotide and the second domain of the fourth polynucleotide willhybridize under appropriate conditions. In some of these aspects, themethod further comprises contacting the target polynucleotide and acomplement of the target polynucleotide with the first polynucleotideand second polynucleotide and the third polynucleotide and fourthpolynucleotide under conditions sufficient to allow hybridization of thefirst domain of the first polynucleotide to the first targetpolynucleotide region of the target polynucleotide, the first domain ofthe second polynucleotide to the second target polynucleotide region ofthe target polynucleotide, the first domain of the third polynucleotideto the first target domain of the complementary strand of the targetpolynucleotide and the first domain of the fourth polynucleotide to thesecond complement target polynucleotide region and extending the firstdomains (i.e., priming domains) of the first and fourth polynucleotideswith a DNA polymerase under conditions which permit extension of thefirst polynucleotide and the third polynucleotide.

In some aspects, a blocking group as described herein above is attachedto the second polynucleotide and/or the fourth polynucleotide at their3′ ends which blocks extension by an enzyme that is capable ofsynthesizing a nucleic acid. Blocking groups useful in the practice ofthe methods include but are not limited to a 3′ phosphate group, a 3′amino group, a dideoxy nucleotide, and inverted deoxythymidine (dT).

In various embodiments, the target polynucleotide, the complement of thetarget polynucleotide or both has a secondary structure that isdenatured by hybridization of the first domain of the secondpolynucleotide and/or the first domain of the fourth polynucleotide to atarget polynucleotide.

In an embodiment, the polynucleotide combinations are contemplated foruse in PCR as depicted in FIG. 7a-f (Schemes 4-9). In Scheme 4, PrimersA (i.e., the first polynucleotide as described herein) and B (i.e., thethird polynucleotide as described herein) are used on opposite strandsof the target polynucleotide, which are depicted as the W-(Watson) andC-(Crick) strands. As is shown in FIG. 7f (Scheme 9), following step 12only the Primers A and B are required for amplification of the targetpolynucleotide.

One of ordinary skill in the art will recognize that the polynucleotidecombinations of the present invention can be used to prime either one orboth ends of a given PCR amplicon. As used herein, an “amplicon” isunderstood to mean a portion of a polynucleotide that has beensynthesized using amplification techniques. It is contemplated that anyof the methods of the present invention that comprise more than onepolynucleotide combination may utilize any combination of standardprimer and polynucleotide combination, provided at least one of theprimers is a polynucleotide combination as described herein.

B. Simple Second Strand Synthesis

In another embodiment, a method of amplifying a target polynucleotide isprovided using the first and second polynucleotides comprisingcontacting the target polynucleotide with the first and secondpolynucleotides disclosed herein under conditions sufficient to allowhybridization of the first domain of the first polynucleotide to thefirst target polynucleotide region of the target polynucleotide and thefirst domain of the second polynucleotide to the second targetpolynucleotide region of the target polynucleotide, and extending thefirst domain (i.e., priming domain) of the first polynucleotide with aDNA polymerase under conditions which permit extension of the firstdomain of the first polynucleotide. In some aspects, the firstpolynucleotide (with associated polynucleotide product extendedtherefrom) and second polynucleotide are then denatured from the targetpolynucleotide and another set of first and second polynucleotides areallowed to hybridize to a target polynucleotide.

In one aspect, the first polynucleotide and the second polynucleotidehybridize sequentially to the target polynucleotide. In another aspect,the first domain of the first polynucleotide hybridizes to the targetbefore the first domain of the second polynucleotide hybridizes to thetarget polynucleotide. In yet another aspect, the first domain of thesecond polynucleotide hybridizes to the target polynucleotide before thefirst domain of the first polynucleotide hybridizes to the targetpolynucleotide. In another aspect, the first domain of the firstpolynucleotide and the first domain of the second polynucleotidehybridize to the target polynucleotide concurrently.

In various embodiments, the target polynucleotide includes but is notlimited to chromosomal DNA, genomic DNA, plasmid DNA, cDNA, RNA, asynthetic polynucleotide, a single stranded polynucleotide, or a doublestranded polynucleotide. In one aspect, the target is a double strandedpolynucleotide and the first domain of the first polynucleotide and thefirst domain of the second polynucleotide hybridize to the same strandof the double stranded target polynucleotide. In another aspect, thesecond domain of the first polynucleotide and the second domain of thesecond polynucleotide hybridize prior to hybridization of the firstpolynucleotide and the second polynucleotide to the targetpolynucleotide.

In an embodiment, the first polynucleotide and the second polynucleotidehybridize to the target polynucleotide concurrently and the thirdpolynucleotide and the fourth polynucleotide hybridize to the complementof the target polynucleotide concurrently, the first polynucleotide andthe second polynucleotide hybridizing to the target polynucleotide atthe same time that the third polynucleotide and the fourthpolynucleotide hybridize to the complement of the target polynucleotide.

In another embodiment, the first polynucleotide, the secondpolynucleotide, the third polynucleotide and the fourth polynucleotidedo not hybridize to the target polynucleotide and the complement of thetarget polynucleotide at the same time.

In yet another embodiment, the second domain of the first polynucleotideand the second domain of the second polynucleotide hybridize prior tohybridizing to the target polynucleotide. In another embodiment, thesecond domain of the third polynucleotide and the second domain of thefourth polynucleotide hybridize prior to hybridizing to the complementof the target polynucleotide.

In an embodiment, the second domain of the first polynucleotide and thesecond domain of the second polynucleotide hybridize prior tohybridizing to the target polynucleotide and the second domain of thethird polynucleotide and the second domain of the fourth polynucleotidehybridize prior to hybridizing to the complement of the targetpolynucleotide.

In another embodiment, the target polynucleotide contains a mutation inthe region to which the first domain of the first polynucleotidehybridizes to the target polynucleotide. In some embodiments, the targetpolynucleotide is fully complementary in the region to which the firstdomain of the first polynucleotide hybridizes to the targetpolynucleotide. In some embodiments, the non-target polynucleotide isnot fully complementary in the region to which the first domain of thefirst polynucleotide hybridizes to the non-target polynucleotide. Inanother embodiment, the target polynucleotide contains a mutation in theregion to which the first domain of the third polynucleotide hybridizesto the target polynucleotide. In some embodiments, the targetpolynucleotide is fully complementary in the region to which the thirddomain of the first polynucleotide hybridizes to the targetpolynucleotide. In some embodiments, the non-target polynucleotide isnot fully complementary in the region to which the third domain of thefirst polynucleotide hybridizes to the non-target polynucleotide. Insome aspects, the mutation is a destabilizing mutation. In relatedaspects, the destabilizing mutation prevents extension of the firstpolynucleotide, or the third polynucleotide, or both.

C. Multiplexing

In an embodiment, the extension by an enzyme that is capable ofsynthesizing a nucleic acid is a multiplex extension, the first domainof the first polynucleotide having the property of hybridizing to morethan one region in the target polynucleotide. In a related embodiment,the extension by an enzyme that is capable of synthesizing a nucleicacid is a multiplex extension, the first domain of the thirdpolynucleotide having the property of hybridizing to more than one locusin the target polynucleotide.

In related embodiments, multiplex PCR is performed using at least twopolynucleotide primers to amplify more than one polynucleotide product.In some aspects of these embodiments, each polynucleotide primer usedfor multiplex PCR is a polynucleotide combination as disclosed herein.In other aspects, at least one polynucleotide primer used for multiplexPCR is a polynucleotide combination as disclosed herein.

In another embodiment, multiplex PCR is performed using multiple fixerpolynucleotides and are directed against genomic repeated sequences. Inanother embodiment, the fixer polynucleotides are comprised of randomsequences. In some of these aspects, multiple fixer polynucleotidesrefers to about 10 polynucleotide sequences. In other aspects, multiplefixer polynucleotides refers to about 15, about 20, about 25, about 30,about 35, about 40, about 45, about 50, about 55, about 60, about 65,about 70, about 75, about 80, about 85, about 90, about 95, about 100,about 150, about 200, about 250, about 300, about 350, about 400, about450, about 500, about 550, about 600, about 650, about 700, about 750,about 800, about 850, about 900, about 950, about 1000 or morepolynucleotide sequences. These fixer polynucleotide sequences wouldprovide a multitude of “fixed” locations in the genome to which amultitude of primer polynucleotides could then bind, taking advantage ofthe unique complementary polynucleotide sequences present in both theprimer and fixer polynucleotides as described herein.

D. Real-Time PCR

Primer combinations with a standard three-way junction are useful forreal-time PCR. Analysis and quantification of rare transcripts,detection of pathogens, diagnostics of rare cancer cells with mutations,or low levels of aberrant gene methylation in cancer patients are theproblems that can be solved by improved real-time PCR assays thatcombine high sensitivity and specificity of target amplification, highspecificity of target detection, the ability to selectively amplify anddetect a small number of cancer-specific mutant alleles or abnormallymethylated promoters in the presence of thousands of copies of normalDNA, analysis and quantification of low copy number RNA transcripts,detection of fluorescence traces the ability to multiplex 4-5 differenttargets in one assay to maximally utilize capabilities of currentreal-time thermal cyclers. A fluorophore is positioned at the 5′ end ofthe primer polynucleotide, and a quencher is positioned at the 3′ end ofthe fixer polynucleotide. In this arrangement, no fluorescence isdetected when the primer and fixer polynucleotides are hybridized (sincethe fluorophore is positioned adjacent to the quencher). However,following extension of the primer polynucleotide during PCR, the primerpolynucleotide and fixer polynucleotide will become separated during thedenaturation phase of PCR, thus creating distance between thefluorophore and the quencher and resulting in a detectable fluorescentsignal.

Primer combinations with a four-way junction can also be used forreal-time PCR. The primer polynucleotide is labeled with a fluorophoreon its 5′ end, and the staple is labeled with a quencher on its 3′ end.The fixer polynucleotide is unlabeled. Since the second domain regionsof both the primer and fixer polynucleotides are unique, the staplepolynucleotide can be used as a “universal” polynucleotide (FIG. 10;Scheme 10).

Primer combinations with a two-way junction and a probe polynucleotidecan also be used for real-time PCR. The probe polynucleotide is labeledwith a fluorophore on its 5′ end, a quencher on its 3′ end, and in someembodiments, an additional internal quencher. When the firstpolynucleotide is extended by a polymerase with 5′ to 3′ exonucleaseactivity, such as Taq polymerase, the label is cleaved and is no longerquenched, resulting in increased signal from the label. In someembodiments, the probe polynucleotide is a molecular beacon probe. Inshort, a molecular beacon probe is comprised of a nucleotide sequencewith bases on its 5′ and 3′ ends that are complementary and form ahairpin structure in the absence of a target polynucleotide. Themolecular beacon probe also comprises a quencher at its 3′ end (or 5′end) and a fluorescent label at its 5′ end (or 3′end) such that there isno detectable signal from the label when the target polynucleotide isnot present. The molecular beacon probe also comprises a sequence thatis complementary to the target polynucleotide such that, in the presenceof the target, hybridization of the probe to the target polynucleotidecauses the dissociation of the hairpin structure and loss of quenching,resulting in a detectable fluorescent signal.

Primer combinations with a two-way junction and a blocker polynucleotidecan also be used in combination for real-time PCR. The primerpolynucleotide (i.e., “first polynucleotide”) is labeled with afluorophore on its 5′ end, and the fixer polynucleotide (i.e., “secondpolynucleotide”) is labeled with a quencher on its 3′ end. The blockerpolynucleotide is complementary to a target polynucleotide regionlocated immediately 5′ of the first target polynucleotide region(depicted in FIG. 4). In some embodiments, the blocker polynucleotideoverlaps with the first domain of the first polynucleotide. In otherwords, the nucleotide(s) at the 3′ end of the first polynucleotide andthe nucleotide(s) at the 5′ end of the blocker polynucleotide would becomplementary to the same nucleotide(s) of the target polynucleotide. Inrelated embodiments, the nucleotide(s) at the 3′ end of the firstpolynucleotide and the nucleotide(s) at the 5′ end of the blockerpolynucleotide are different. In these embodiments, the nucleotide(s) atthe 3′ end of the first polynucleotide would hybridize to the targetpolynucleotide when it is complementary to the target polynucleotide atthe appropriate position(s), thus allowing for extension of the firstpolynucleotide under the appropriate conditions (see FIG. 4a ).Following extension of the primer polynucleotide during PCR, the primerpolynucleotide and fixer polynucleotide will become separated during thedenaturation phase of PCR, thus creating distance between thefluorophore and the quencher and resulting in a detectable fluorescentsignal. In related embodiments, the nucleotide at the 5′ end of theblocker polynucleotide would hybridize to the non-target polynucleotidewhen it is complementary to the non-target polynucleotide at theappropriate position, thus blocking extension of the firstpolynucleotide. (see FIG. 4b ). In this arrangement, no fluorescence isdetected when the primer and fixer polynucleotides are hybridized (sincethe fluorophore is positioned adjacent to the quencher). In variousembodiments, the nucleotide at the 3′ end of the blocker polynucleotideis modified to prevent extension by a polymerase. This system allows fordetection of, for example, single nucleotide polymorphisms with greatsensitivity and specificity.

Primer combinations with two-way junctions, blocker polynucleotides, andprobe polynucleotides are also used in combination for real-time PCR. Inrelated embodiments, the first polynucleotide used in this combinationcomprises a modified nucleic acid as the nucleotide at its 3′ end andthe blocker polynucleotide comprises a modified nucleic acid as thenucleotide at its 5′ end. In some embodiments, the modified nucleic acidis a locked nucleic acid.

In some aspects, the above embodiments further comprise a reverse primerpolynucleotide. The reverse primer is complementary to a region in thepolynucleotide created by extension of the first polynucleotide. SeeFIG. 8. As is apparent, in some embodiments the reverse primer is alsocomplementary to the complementary strand of the target polynucleotidewhen the target polynucleotide is one strand of a double-strandedpolynucleotide. Inclusion of a reverse primer allows for amplificationof the target polynucleotide. In various aspect, the reverse primer is a“simple” primer wherein the sequence of the reverse primer is designedto be sufficiently complementary over its entire length to hybridize toa target sequence over the entire length of the primer. A simple primerof this type is in one aspect, 100% complementary to a target sequence,however, it will be appreciated that a simple primer withcomplementarity of less than 100% is useful under certain circumstancesand conditions.

In other aspect, a reverse primer is a separate polynucleotide primercombination that specifically binds to regions in a sequence produced byextension of a polynucleotide from the first domain of the firstpolynucleotide in a primer pair combination used in a first reaction.

In various aspects, the methods described herein provide a change insequence detection from a sample with a non-target polynucleotidecompared to sequence detection from a sample with a targetpolynucleotide. In some aspects, the change is an increase in detectionof a target polynucleotide in a sample compared to sequence detectionfrom a sample with a non-target polynucleotide. In some aspects, thechange is a decrease in detection of a target polynucleotide in a samplecompared to sequence detection from a sample with a non-targetpolynucleotide.

Due to the increased specificity of the polynucleotides describedherein, real-time PCR can be performed in the presence of SYBR green dyeto achieve a specificity that is equivalent to that achieved usingTaqMan, molecular beacon probes or Scorpion primers but at a greatlyreduced cost.

In one embodiment, the primer polynucleotide (i.e., “firstpolynucleotide”) is labeled with a fluorescent molecule at its 5′ endand a second quenching polynucleotide (i.e., “universal quencherpolynucleotide”) that is labeled at its 3′ end with a quencher are bothhybridized to the second domain of the fixer polynucleotide (i.e.,“second polynucleotide”), which comprises a blocking group at its 3′ endto prevent extension from a DNA polymerase. This complex has nofluorescence in this state but will fluoresce when the complex isdisplaced (denatured) following extension of the primer polynucleotideby a DNA polymerase.

In another embodiment, the primer polynucleotide (i.e., “firstpolynucleotide”) comprising a fluorophore at its 5′ end is hybridized toa fixer polynucleotide (i.e., “second polynucleotide”) comprising aquencher at its 3′end. The complex has no fluorescence when hybridized,but will fluoresce when the complex is displaced (denatured) followingextension of the primer polynucleotide by a DNA polymerase. In anotheraspect of the method, multiplex real-time PCR is performed using twosets of polynucleotide combinations, wherein one polynucleotide in eachprimer set is labeled with a fluorophore, and the two fluorophores aredistinguishable from each other.

In another embodiment, the primer polynucleotide (i.e., “firstpolynucleotide”) comprises a fluorophore, a quencher on its 3′ end, andthese two labels are separated by a stretch of RNA or RNA/DNAoligonucleotides (i.e., “probe polynucleotide”) (see FIG. 9). In someaspects, the probe polynucleotide further comprises an internal Zenquencher. In some aspects, a fluorescent signal is generated uponcreation and degradation of the RNA/DNA hybrid by a thermostable RNase Hand release of a free fluorophore (or quencher) into solution.

FIG. 9A depicts a fluorophore-quencher labeled first polynucleotide(Primer A) with a non-specific RNA linker and a typical secondpolynucleotide (Fixer A). In certain embodiments, the firstpolynucleotide (P) comprises a 5′ label followed by an RNA sequence,followed by a quencher, followed by a sequence typical of firstpolynucleotides (P) described herein. In other embodiments, the firstpolynucleotide (P) comprises a 5′ quencher followed by an RNA sequence,followed by a label, followed by a sequence typical of firstpolynucleotides (P) described herein. FIG. 9B illustrates the use of thecombination depicted in FIG. 9A in PCR. When the strand opposite P isgenerated, cleavage of the RNA-DNA hybrid by RNase H releases thefluorophore (or quencher) and a fluorescent signal is detected.

FIG. 9C depicts a fluorophore-quencher labeled first polynucleotide(Primer A) with a site-specific RNA-DNA linker and a typical secondpolynucleotide (Fixer A). In certain embodiments, the firstpolynucleotide (P) comprises a 5′ label followed by an RNA sequence thatis complementary to a sequence downstream of P, followed by a quencher,followed by a sequence typical of first polynucleotides (P) describedherein. In certain embodiments, the first polynucleotide (P) comprises a5′ quencher, followed by an RNA sequence that is complementary to asequence downstream of P, followed by a label, followed by a sequencetypical of first polynucleotides (P) described herein. FIG. 9Dillustrates the use of the combination depicted in FIG. 9C in PCR. Whenthe PCR product comprising Primer A is denatured, the RNA-DNA linkerhybridizes to a region downstream of Primer A, RNase H cleaves theRNA-DNA hybrid and releases the fluorophore and a fluorescent signal isdetected.

In some embodiments, one fixer polynucleotide (i.e., “secondpolynucleotide”) may be used in combination with 2, 3, 4, 5 or moreprimer polynucleotides (i.e., “first polynucleotides”) for simultaneousmultiplex detection of several mutations in one real-time PCR assay.

In another embodiment, a kit is provided comprising, e.g., a packageinsert, a set of four fluorescently labeled universal polynucleotidemolecules, a universal polynucleotide molecule comprising a quencher atits 3′ end, and a DNA ligase with appropriate buffer for assembly of thefluorescently labeled primer polynucleotide. The kit optionally furthercomprises a T₄ polynucleotide kinase and appropriate buffer.

The kit is used to fluorescently label polynucleotides through ligation.In one embodiment, a primer polynucleotide is phosphorylated with T₄polynucleotide kinase and is subsequently hybridized to a fixerpolynucleotide. A third polynucleotide (i.e., a fluorescently labeleduniversal polynucleotide, see above) comprising a fluorophore at its 5′end is likewise hybridized to the fixer polynucleotide. The 3′ end ofthe third polynucleotide is then ligated to the phosphorylated 5′ end ofthe primer polynucleotide, creating a fluorescently-labeled primerpolynucleotide. Finally, a universal polynucleotide comprising aquencher at its 3′ end is hybridized to the fixer polynucleotide,resulting in a polynucleotide complex that has no fluorescence and isready for use in, e.g., a real-time PCR analysis.

E. Primer Extension

The primer compositions disclosed herein can be used in any methodrequiring or utilizing primer extension. For example, primer extensioncan be used to determine the start site of RNA transcription for a knowngene. This technique requires a labeled primer polynucleotidecombination as described herein (usually 20-50 nucleotides in length)which is complementary to a region near the 3′ end of the gene. Thepolynucleotide combination is allowed to anneal to the RNA and reversetranscriptase is used to synthesize complementary (cDNA) to the RNAuntil it reaches the 5′ end of the RNA. By analyzing the product on apolyacrylamide gel, it is possible to determine the transcriptionalstart site, as the length of the sequence on the gel represents thedistance from the start site to the labeled primer.

The advanced polynucleotide technology described herein would overcomeand resolve potential secondary structure encountered in RNA.

F. Isothermal DNA Amplification

Isothermal DNA amplification may be performed as taught in U.S. Pat. No.7,579,153 using the advanced polynucleotide technology described herein.Briefly, isothermal DNA amplification comprises the following steps: (i)providing a double stranded DNA having a hairpin at one end, thepolynucleotide at the other end, and disposed therebetween a promotersequence oriented so that synthesis by an RNA polymerase recognizing thepromoter sequence proceeds in the direction of the hairpin; (ii)transcribing the double stranded DNA with an RNA polymerase thatrecognizes the promoter sequence to form an RNA transcript comprisingcopies of the promoter sequence and the polynucleotide; (iii) generatinga complementary DNA from the RNA transcript; (iv) displacing a 5′ end ofthe RNA transcript from the complementary DNA so that the hairpin isreconstituted; and (v) extending the hairpin to generate the doublestranded DNA containing a reconstituted promoter sequence, the RNApolymerase recognizing the reconstituted promoter sequence andsynthesizing RNA transcripts. In a preferred embodiment, the step ofgenerating includes forming a heteroduplex of said complementary DNA andsaid RNA transcript and wherein said step of displacing includestreating the heteroduplex with a helicase.

G. Fluorescence In Situ Hybridization (FISH)

The advanced polynucleotide technology described herein can also be usedto practice FISH. FISH is a cytogenetic technique used to detect andlocalize the presence or absence of specific DNA sequences onchromosomes. FISH uses fluorescent probes that bind to only those partsof the chromosome with which they show a high degree of sequencesimilarity. Fluorescence microscopy can be used to find out where thefluorescent probe bound to the chromosomes. FISH is often used forfinding specific features in DNA for use in genetic counseling,medicine, and species identification. FISH can also be used to detectand localize specific mRNAs within tissue samples. In this context, itcan help define the spatial-temporal patterns of gene expression withincells and tissues.

H. Ligation Probes

The advanced polynucleotide technology described herein can also be usedto practice multiplex PCR using ligation probes. Ligation probe methodsare known to those of skill in the art. Briefly, ligation probes consistof two separate oligonucleotides, each containing a PCR primer sequence.It is only when these two hemi probes are both hybridized to theiradjacent targets that they can be ligated. Only ligated probes will beamplified exponentially in a PCR. The number of probe ligation productstherefore depends on the number of target sequences in the sample.

In some embodiments, two ligation probes are separated by about 1 toabout 500 nucleotides, and prior to ligation the first probe is extendedby a DNA thermostable polymerase lacking strand-displacement activity. ADNA thermostable polymerase lacking strand-displacement activityincludes, but is not limited to, a Pfu polymerase. When the extendedstrand reaches the 5′-phosphate group of the second ligation probe,polymerization stops and a nick is created. This nick can be sealed by athermostable ligase present in the reaction mixture, allowing for theentire reaction to occur in a single-reaction format.

I. Next Generation Sequencing (NGS)

The polynucleotide combinations of the present invention may also beused in NGS applications. Instead of sequencing by sequential ligationof DNA probes, the primer combinations disclosed herein can be used insequential hybridization without ligation, as shown in FIG. 15. For areview of NGS technology, see Morozova et al., Genomics 92(5): 255-64,2008, incorporated herein by reference in its entirety. NGS is readilyunderstood and practiced by those of ordinary skill in the art andexemplified in one embodiment as set out below in Example 2.

X. Enzymes

In some aspects of any of the methods, the extension is performed by anenzyme that is capable of synthesizing a nucleic acid is quantitated inreal-time. The enzymes useful in the practice of the invention includebut are not limited to a DNA polymerase (which can include athermostable DNA polymerase, e.g., a Taq DNA polymerase), RNApolymerase, and reverse transcriptase. Non-limiting examples of enzymesthat may be used to practice the present invention include but are notlimited to Deep VentR™ DNA Polymerase, LongAmp™ Taq DNA Polymerase,Phusion™ High-Fidelity DNA Polymerase, Phusion™ Hot Start High-FidelityDNA Polymerase, VentR® DNA Polymerase, DyNAzyme™ II Hot Start DNAPolymerase, Phire™ Hot Start DNA Polymerase, Phusion™ Hot StartHigh-Fidelity DNA Polymerase, Crimson LongAmp™ Taq DNA Polymerase,DyNAzyme™ EXT DNA Polymerase, LongAmp™ Taq DNA Polymerase, Phusion™High-Fidelity DNA Polymerase, Phusion™ Hot Start High-Fidelity DNAPolymerase, Taq DNA Polymerase with Standard Taq (Mg-free) Buffer, TaqDNA Polymerase with Standard Taq Buffer, Taq DNA Polymerase withThermoPol II (Mg-free) Buffer, Taq DNA Polymerase with ThermoPol Buffer,Crimson Taq™ DNA Polymerase, Crimson Taq™ DNA Polymerase with (Mg-free)Buffer, Phire™ Hot Start DNA Polymerase, Phusion™ High-Fidelity DNAPolymerase, VentR® DNA Polymerase, VentR® (exo-) DNA Polymerase, Phire™Hot Start DNA Polymerase, Phusion™ High-Fidelity DNA Polymerase,Phusion™ Hot Start High-Fidelity DNA Polymerase, Hemo KlenTaq™, DeepVentR™ (exo-) DNA Polymerase, Deep VentR™ DNA Polymerase, DyNAzyme™ EXTDNA Polymerase, Hemo KlenTaq™, LongAmp™ Taq DNA Polymerase, Phusion™High-Fidelity DNA Polymerase, Prot® Script® AMV First Strand cDNASynthesis Kit, Prot® Script® M-MuLV First Strand cDNA Synthesis Kit, BstDNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, Taq DNAPolymerase with ThermoPol Buffer, 9° Nm DNA Polymerase, Crimson Taq™ DNAPolymerase, Crimson Taq™ DNA Polymerase with (Mg-free) Buffer, DeepVentR™ (exo-) DNA Polymerase, Deep VentR™ DNA Polymerase, DyNAzyme™ EXTDNA Polymerase, DyNAzyme™ II Hot Start DNA Polymerase, Hemo KlenTaq™,Phusion™ High-Fidelity DNA Polymerase, Phusion™ Hot Start High-FidelityDNA Polymerase, Sulfolobus DNA Polymerase IV, Therminator™ y DNAPolymerase, Therminator™ DNA Polymerase, Therminator™ II DNA Polymerase,Therminator™ III DNA Polymerase, VentR® DNA Polymerase, VentR® (exo-)DNA Polymerase, Bsu DNA Polymerase, Large Fragment, DNA Polymerase I (E.coli), DNA Polymerase I, Large (Klenow) Fragment, Klenow Fragment (3′→5′exo-), phi29 DNA Polymerase, T4 DNA Polymerase, T7 DNA Polymerase(unmodified), Terminal Transferase, Reverse Transcriptases and RNAPolymerases, E. coli Poly(A) Polymerase, AMV Reverse Transcriptase,M-MuLV Reverse Transcriptase, phi6 RNA Polymerase (RdRP), Poly(U)Polymerase, SP6 RNA Polymerase, and T7 RNA Polymerase.

XI. Labels

In some aspects, the first polynucleotide comprises a label. In otheraspects, any polynucleotide used in the methods described hereincomprises a label. In some of these aspects the label is fluorescent.Methods of labeling oligonucleotides with fluorescent molecules andmeasuring fluorescence are well known in the art. Fluorescent labelsuseful in the practice of the invention include but are not limited to1,8-ANS (1-Anilinonaphthalene-8-sulfonic acid),1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 5-(and-6)-Carboxy-2′,7′-dichlorofluorescein pH 9.0, 5-FAM pH 9.0, 5-ROX(5-Carboxy-X-rhodamine, triethylammonium salt), 5-ROX pH 7.0, 5-TAMRA,5-TAMRA pH 7.0, 5-TAMRA-MeOH, 6 JOE,6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0, 6-Carboxyrhodamine 6G pH7.0, 6-Carboxyrhodamine 6G, hydrochloride, 6-HEX, SE pH 9.0, 6-TET, SEpH 9.0, 7-Amino-4-methylcoumarin pH 7.0, 7-Hydroxy-4-methylcoumarin,7-Hydroxy-4-methylcoumarin pH 9.0, Alexa 350, Alexa 405, Alexa 430,Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 568, Alexa 594, Alexa647, Alexa 660, Alexa 680, Alexa 700, Alexa Fluor 430 antibody conjugatepH 7.2, Alexa Fluor 488 antibody conjugate pH 8.0, Alexa Fluor 488hydrazide-water, Alexa Fluor 532 antibody conjugate pH 7.2, Alexa Fluor555 antibody conjugate pH 7.2, Alexa Fluor 568 antibody conjugate pH7.2, Alexa Fluor 610 R-phycoerythrin streptavidin pH 7.2, Alexa Fluor647 antibody conjugate pH 7.2, Alexa Fluor 647 R-phycoerythrinstreptavidin pH 7.2, Alexa Fluor 660 antibody conjugate pH 7.2, AlexaFluor 680 antibody conjugate pH 7.2, Alexa Fluor 700 antibody conjugatepH 7.2, Allophycocyanin pH 7.5, AMCA conjugate, Amino Coumarin, APC(allophycocyanin), Atto 647, BCECF pH 5.5, BCECF pH 9.0, BFP (BlueFluorescent Protein), BO-PRO-1-DNA, BO-PRO-3-DNA, BOBO-1-DNA,BOBO-3-DNA, BODIPY 650/665-X, MeOH, BODIPY FL conjugate, BODIPY FL,MeOH, Bodipy R6G SE, BODIPY R6G, MeOH, BODIPY TMR-X antibody conjugatepH 7.2, Bodipy TMR-X conjugate, BODIPY TMR-X, MeOH, BODIPY TMR-X, SE,BODIPY TR-X phallacidin pH 7.0, BODIPY TR-X, MeOH, BODIPY TR-X, SE,BOPRO-1, BOPRO-3, Calcein, Calcein pH 9.0, Calcium Crimson, CalciumCrimson Ca2+, Calcium Green, Calcium Green-1 Ca2+, Calcium Orange,Calcium Orange Ca2+, Carboxynaphthofluorescein pH 10.0, Cascade Blue,Cascade Blue BSA pH 7.0, Cascade Yellow, Cascade Yellow antibodyconjugate pH 8.0, CFDA, CFP (Cyan Fluorescent Protein), CI-NERF pH 2.5,CI-NERF pH 6.0, Citrine, Coumarin, Cy 2, Cy 3, Cy 3.5, Cy 5, Cy 5.5,CyQUANT GR-DNA, Dansyl Cadaverine, Dansyl Cadaverine, MeOH, DAPI,DAPI-DNA, Dapoxyl (2-aminoethyl) sulfonamide, DDAO pH 9.0, Di-8 ANEPPS,Di-8-ANEPPS-lipid, DiI, DiO, DM-NERF pH 4.0, DM-NERF pH 7.0, DsRed,DTAF, dTomato, eCFP (Enhanced Cyan Fluorescent Protein), eGFP (EnhancedGreen Fluorescent Protein), Eosin, Eosin antibody conjugate pH 8.0,Erythrosin-5-isothiocyanate pH 9.0, Ethidium Bromide, Ethidiumhomodimer, Ethidium homodimer-1-DNA, eYFP (Enhanced Yellow FluorescentProtein), FDA, FITC, FITC antibody conjugate pH 8.0, FlAsH, Fluo-3,Fluo-3 Ca2+, Fluo-4, Fluor-Ruby, Fluorescein, Fluorescein 0.1 M NaOH,Fluorescein antibody conjugate pH 8.0, Fluorescein dextran pH 8.0,Fluorescein pH 9.0, Fluoro-Emerald, FM 1-43, FM 1-43 lipid, FM 4-64, FM4-64, 2% CHAPS, Fura Red Ca2+, Fura Red, high Ca, Fura Red, low Ca,Fura-2 Ca2+, Fura-2, high Ca, Fura-2, no Ca, GFP (S65T), HcRed, Hoechst33258, Hoechst 33258-DNA, Hoechst 33342, Indo-1 Ca2+, Indo-1, Ca free,Indo-1, Ca saturated, JC-1, JC-1 pH 8.2, Lissamine rhodamine,LOLO-1-DNA, Lucifer Yellow, CH, LysoSensor Blue, LysoSensor Blue pH 5.0,LysoSensor Green, LysoSensor Green pH 5.0, LysoSensor Yellow pH 3.0,LysoSensor Yellow pH 9.0, LysoTracker Blue, LysoTracker Green,LysoTracker Red, Magnesium Green, Magnesium Green Mg2+, MagnesiumOrange, Marina Blue, mBanana, mCherry, mHoneydew, MitoTracker Green,MitoTracker Green FM, MeOH, MitoTracker Orange, MitoTracker Orange,MeOH, MitoTracker Red, MitoTracker Red, MeOH, mOrange, mPlum, mRFP,mStrawberry, mTangerine, NBD-X, NBD-X, MeOH, NeuroTrace 500/525, greenfluorescent Nissl stain-RNA, Nile Blue, EtOH, Nile Red, Nile Red-lipid,Nissl, Oregon Green 488, Oregon Green 488 antibody conjugate pH 8.0,Oregon Green 514, Oregon Green 514 antibody conjugate pH 8.0, PacificBlue, Pacific Blue antibody conjugate pH 8.0, Phycoerythrin, PO-PRO-1,PO-PRO-1-DNA, PO-PRO-3, PO-PRO-3-DNA, POPO-1, POPO-1-DNA, POPO-3,Propidium Iodide, Propidium Iodide-DNA, R-Phycoerythrin pH 7.5, ReAsH,Resorufin, Resorufin pH 9.0, Rhod-2, Rhod-2 Ca2+, Rhodamine, Rhodamine110, Rhodamine 110 pH 7.0, Rhodamine 123, MeOH, Rhodamine Green,Rhodamine phalloidin pH 7.0, Rhodamine Red-X antibody conjugate pH 8.0,Rhodaminen Green pH 7.0, Rhodol Green antibody conjugate pH 8.0,Sapphire, SBFI-Na+, Sodium Green Na+, Sulforhodamine 101, SYBR Green I,SYPRO Ruby, SYTO 13-DNA, SYTO 45-DNA, SYTOX Blue-DNA,Tetramethylrhodamine antibody conjugate pH 8.0, Tetramethylrhodaminedextran pH 7.0, Texas Red-X antibody conjugate pH 7.2, TO-PRO-1-DNA,TO-PRO-3-DNA, TOTO-1-DNA, TOTO-3-DNA, TRITC, X-Rhod-1 Ca2+,YO-PRO-1-DNA, YO-PRO-3-DNA, YOYO-1-DNA, and YOYO-3-DNA.

Other labels besides fluorescent molecules can be used, such aschemiluminescent molecules, which will give a detectable signal or achange in detectable signal upon hybridization, and radioactivemolecules.

In some embodiments, the second polynucleotide comprises a quencher thatattenuates the fluorescence signal of a label. In other embodiments, thefourth polynucleotide comprises a quencher that attenuates thefluorescence signal of a label. Quenchers contemplated for use inpractice of the methods of the invention include but are not limited toBlack Hole Quencher 1, Black Hole Quencher-2, Iowa Black FQ, Iowa BlackRQ, Zen quencher, and Dabcyl. G-base.

XII. Modified Polynucleotide Combinations

Partially double-stranded primer combinations with modified propertiescan be formed: (1) By two polynucleotides such as a basic primerpolynucleotide and a basic fixer polynucleotide; (2) By twopolynucleotides such as a modified primer polynucleotide and a basicfixer polynucleotide; (3) By two polynucleotides such as a modifiedprimer polynucleotide and a modified fixer polynucleotide; (4) By threepolynucleotides such as a basic primer polynucleotide, a basic fixerpolynucleotide and an anti-primer polynucleotide; (5) By threepolynucleotides such as a modified primer polynucleotide, a basic fixerpolynucleotide and an anti-primer polynucleotide.

Partially double-stranded polynucleotides can initiate hybridization togenomic DNA only by its single-stranded region that can be locatedwithin the linear portion of a polynucleotide or within the loop region.

Non-complete sequence complementarity (including a single nucleotidemismatch) within the single-stranded region of the polynucleotidesignificantly reduces the efficiency of hybridization by slowing downthe initiation of hybridization.

Non-complete sequence complementarity within the double-stranded regionof the polynucleotide should interfere with strand-displacementhybridization and binding of the polynucleotide to the templatepolynucleotide.

Modified polynucleotides that are more sensitive to changes in templatepolynucleotide sequence than the basic polynucleotides can be used fordevelopment of more specific PCR-based diagnostic assays and for moresensitive PCR detection of rare DNA mutations in, e.g., cancer tissues.

FIG. 11 (Scheme 11) depicts three examples utilizing basic primer andfixer polynucleotides with a non-covalently attached anti-primer (AP).

In another embodiment, polynucleotides may be constructed with a basicprimer polynucleotide (“PO”) and a modified fixer polynucleotidestructure (“F1 through F4”) (see FIG. 12; Scheme 12). The modifiedfixers are shown on the left, with the complete polynucleotidecombinations (basic primer and modified fixer polynucleotides) shown onthe right.

The modified polynucleotide combinations comprising the stem loopstructures (1 through 4, Scheme 12) may be utilized to provide anincreased level of specificity when binding to a templatepolynucleotide. In these aspects, a single-stranded region (e.g., in aloop structure) hybridizes to a template polynucleotide. Thishybridization, if 100% complementary in sequence, will efficientlydisplace to a fully complementary stem portion of the fixerpolynucleotide. In a related aspect, a primer polynucleotide can thenhybridize to the fully-hybridized fixer polynucleotide. Any mutationswithin the single-stranded loop or the double-stranded stem regions ofthe modified fixer polynucleotide will reduce its hybridizationefficiency and, as a result, the priming efficiency of thepolynucleotide combination.

In another embodiment, polynucleotide combinations may be constructedwith a modified primer polynucleotide (“P1”) and a basic fixerpolynucleotide (“F”) (see FIG. 13a ; Scheme 13). In Scheme 13, themodified primer polynucleotide is shown on the left, with the completepolynucleotide combination (modified primer polynucleotide and basicfixer polynucleotide) shown on the right. In this embodiment, thesingle-stranded DNA linker (comprised of, e.g., poly dT) should be longenough to allow the 5′ segment of primer 1 to hybridize to its 3′segment.

In another embodiment, polynucleotide combinations may be constructedwith a modified primer polynucleotide (Primer 1, P1) and a modifiedfixer polynucleotide (see Scheme 14). Two such examples are shown inFIG. 12b (Scheme 14), shown with modified fixer polynucleotides F1 andF4, from FIG. 12 (Scheme 12).

In another embodiment, polynucleotide combinations with a modifiedprimer structure further comprise non-covalently attached anti-primers.In one aspect, polynucleotide combinations may be formed that comprise amodified primer polynucleotide, a basic fixer polynucleotide and ananti-primer. In various aspects, the anti-primer may be hybridized todifferent regions of the polynucleotide combination (see FIG. 12c ;Scheme 15).

Anti-primer polynucleotides serve to further increase the specificity ofbinding of the first domain of the fixer polynucleotide to the templatepolynucleotide. Here, only 100% complementary template polynucleotideregions will efficiently hybridize to the short polynucleotide regionthat is not covered by the anti-primer. As the first domain of the fixerpolynucleotide hybridizes to the template polynucleotide, it willdisplace the anti-primer if the template region is 100% complementary tothe fixer polynucleotide. This provides an extra level of specificityversus the fixer polynucleotide alone.

The references cited herein throughout, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are all specifically incorporated herein by reference.

EXAMPLES

A person of skill in the art will appreciate that when primers or primercombinations are referred to as being in “forward” or “reverse”orientations, these designations are arbitrary conventions used indescribing PCR reactions and the structural relationship of the primersand the template. Thus, as is apparent to a person of skill in the art,re-orienting a PCR schematic diagram by flipping it 180° would result in“forward” primers becoming “reverse” primers and “reverse” primersbecoming “forward” primers, and as such, designation of, for example,one primer combination as a forward primer or a reverse primer is not alimitation on the structure or use of that particular primercombination.

Example 1

Basic Single Base Mutation Detection PCR using polynucleotidecombinations of the invention. In one of its most basic forms, PCR isperformed using two polynucleotide combinations. One polynucleotidecombination is the “forward” complex and the other is the “reverse”complex. Each polynucleotide combination is comprised of twopolynucleotides, a primer and a fixer. The primer and fixerpolynucleotides are able to hybridize to each other as well as to atemplate DNA polynucleotide, as depicted in Scheme 2 (FIG. 2).

In the case of mutation detection, the forward and/or reverse primerpolynucleotides contain a sequence that is able to discern a mutant froma wild type sequence in a target DNA polynucleotide. If the primerpolynucleotide sequence is directed to a wild type sequence, then theprimer polynucleotide will only bind efficiently to a wild typetemplate, and vice versa. This result is because the mutant sequencewill differ from the wild type sequence at only a single base position,and an aspect of the polynucleotide combinations described herein isthat the first domain of the primer polynucleotide that hybridizes tothe template polynucleotide is short (5 to 30 nucleotides). This lengthallows for the discrimination of mutant and wild type sequences, sinceif there is a mismatch the primer oligonucleotide binding will beunstable and consequently unable to prime DNA synthesis.

The first step is to assemble the reagents in a reaction vessel. Thereagents comprise the forward and reverse polynucleotide combinationcomplexes, a template DNA polynucleotide, a thermostable DNA polymerase,deoxynucleotide substrates, and a suitable buffer. The PCR is carriedout according to methods well known in the art, using optimizedconditions that are easily determined by those of skill in the art.

Depending on how the primer polynucleotides are designed (i.e., whetherthey are designed to detect a mutant or wild type allele) the resultingPCR products provides information on whether the sample contains amutant or wild type allele (or both).

FIG. 14 (Scheme 16) depicts the two scenarios. In the top scenario, thefirst domain of a primer polynucleotide is 100% complementary to thetemplate DNA polynucleotide and extension occurs, yielding a product.

In the bottom scenario, the first domain of the primer polynucleotidecontains a mismatch relative to the template DNA polynucleotide andextension is blocked due to the instability in the short first domain ofthe primer polynucleotide. This instability will result in a very lowefficiency of PCR and will yield very little or no detectable product.

Example 2

Next Generation Sequencing. Use of a polynucleotide combination toperform next generation sequencing (NGS) is performed without the use ofeither probe ligation or polymerase extension (FIG. 15; Scheme 17).

First, a first fixer polynucleotide and a mixture of four fluorescentlylabeled polynucleotides are hybridized to a polynucleotide template. Thetemplate with bound polynucleotides is then washed and the signal isread.

Next, the fluorescent label is cleaved and the mixture is washed again.Then a mixture of four fluorescently labeled polynucleotides arehybridized to the template polynucleotide, the mixture is washed and thesignal is read again.

The first two steps are repeated until the end of the template isreached. Then the first fixer polynucleotide and all hybridizedpolynucleotides are stripped from the template polynucleotide. This stepis followed by hybridization of a second fixer polynucleotide thathybridizes a single base upstream from the first fixer polynucleotide.

As before, a mixture of four fluorescently labeled polynucleotides arehybridized to the template polynucleotide, the mixture washed, and thesignal read. The fluorescent label is then cleaved, the mixture washed,and the four fluorescently labeled polynucleotides are again allowed tohybridize. The mixture is then washed and the signal read.

These steps are again repeated until the end of the templatepolynucleotide is reached. In this way, the DNA sequence is obtainedwithout the use of a DNA polymerase.

Example 3 Quantitative PCR in the Presence of Staining Dye SYTO 9Materials:

Substrate: Lambda DNA New England Biolabs # N3011S

P10-35 (SEQ ID NO: 4) (i.e., “first polynucleotide”)

F10-23 (SEQ ID NO: 3) (i.e., “second polynucleotide”)

Forward primer 10-15 (SEQ ID NO: 1)

Reverse primer 10-17 (SEQ ID NO: 2)

Taq DNA polymerase New England Biolabs # M0320L

Taq DNA polymerase buffer: 10 mM Tris-HCl, 50 mM KCl pH 8.3 @ 25° C.

dNTPs: Invitrogen #10297018

SYTO® 9 green fluorescent nucleic acid stain Invitrogen # S34854Methods:

Amplification was carried out in triplicate in 25 μl volume aliquots.

Normal amplification reactions consisted of 12.5 μl of 2×Taq DNApolymerase buffer, 3 mM MgCl₂, 200 nM of conventional forward primer10-15 (SEQ ID NO: 1), conventional reverse primer 10-17 (SEQ ID NO: 2),200 μM of dNTPs, 1 unit of Taq polymerase, 0.1 ng of Lambda DNA and 2 uMof SYTO® 9. Amplification was performed in BioRad CFX96 Real Time Systemusing the following thermal cycling profile: one cycle at 94° C. for 2min, followed by 50 cycles at 94° C. for 15 seconds and 66° C. for 1minute 20 seconds.

Primer combination P10-35 and F10-23 amplification mixes consisted of12.5 μl of 2×Taq DNA polymerase buffer, 3 mM MgCl₂, 200 nM ofconventional forward primer 10-15 (SEQ ID NO: 1), 200 nM of P10-35 (SEQID NO: 4), 400 μM of F10-23 (SEQ ID NO: 3), 200 μM of dNTPs, 1 unit ofTaq polymerase, 0.1 ng of Lambda DNA and 2 uM of SYTO® 9. Amplificationparameters were the same as for the normal primer amplification.

Results:

Averaged amplification curves for the reaction with two conventionalprimers, 10-15 (SEQ ID NO: 1) and 10-17 (SEQ ID NO: 2), and the reactionwith forward conventional primer 10-15 (SEQ ID NO: 1), P10-35 (SEQ IDNO: 4), and F10-23 (SEQ ID NO: 3) are shown in FIG. 16. The resultsindicate that the assay containing the P10-35/F10-23 pair performs aswell as the assay containing conventional primers.

Example 4

Quantitative PCR Assay with Fluorophor-Labeled Primer Combination andQuencher-Labeled Fixer

Materials:

Substrate: Lambda DNA New England Biolabs # N3011S

5′-Fluorescein-labeled P10-74 (SEQ ID NO: 10) (i.e., “firstpolynucleotide comprising a label”)

3′-Iowa Black quencher-labeled F10-73 (SEQ ID NO: 9) (i.e., “secondpolynucleotide comprising a quencher”)

Forward primer 10-15 (SEQ ID NO: 1)

Taq DNA polymerase New England Biolabs # M0320L

Taq DNA polymerase buffer: 10 mM Tris-HCl, 50 mM KCl pH 8.3 @ 25° C.

Vent (exo-) DNA polymerase New England Biolabs # M0257L

dNTPs: Invitrogen #10297018 Methods:

Amplification reaction with P10-74 contained 12.5 μl of 2×Taq DNApolymerase buffer, 3 mM MgCl₂, 200 nM of conventional forward primer10-15 (SEQ ID NO: 1), 200 nM of P10-74 (SEQ ID NO: 10), and 400 nM ofF10-73 (SEQ ID NO: 9), 200 μM of dNTPs, 1 unit of Taq polymerase and 0.1ng of Lambda DNA. Amplification was performed in BioRad CFX96 Real TimeSystem using the following thermal cycling profile: one cycle at 94° C.for 2 minutes, followed by 50 cycles at 94° C. for 15 seconds and 66° C.for 1 minute 20 seconds and “read” cycle for 10 seconds at 60° C. Somereactions were also supplied with 0.2 units of Vent (exo-) polymerase.

Results:

Amplification real-time PCR curves are shown in FIG. 17. Assay withfluorophor-labeled P10-74 generated a reasonably strong signal that wasslightly improved by adding Vent (exo-) polymerase.

Conclusions:

qPCR assay with the P10-74/F10-73 pair with label and quencher,respectively, represents a novel tool for diagnostic applications.

Example 5

qPCR Assay with a Fluorophor-Labeled Primer Combination and UniversalQuencher

Materials:

Substrate: Lambda DNA New England Biolabs # N3011S

P10-104 with 3′ fluorophore-label and protected bonds (SEQ ID NO: 13)(i.e., “first polynucleotide comprising a label”),

F10-79 (SEQ ID NO: 11) (i.e., “second polynucleotide”),

Quencher oligonucleotide 10-80 (SEQ ID NO: 12) (i.e., “universalquencher polynucleotide”),

Forward primer 10-15 (SEQ ID NO: 1),

Taq DNA polymerase New England Biolabs # M0320L

Taq DNA polymerase buffer: 10 mM Tris-HCl, 50 mM KCl pH 8.3 @ 25° C.

Vent (exo-) DNA polymerase New England Biolabs # M0257L

dNTPs: Invitrogen #10297018

Methods:

Amplification was carried out in triplicate with 25 μl volume aliquotscontaining 12.5 μl of 2×Taq DNA polymerase buffer, 3 mM MgCl₂, 200 nM ofconventional forward primer 10-15 (SEQ ID NO: 1), 200 nM of P10-104 (SEQID NO: 13), 400 nM of F10-79 (SEQ ID NO: 11), 600 nM of Quencher 10-80(SEQ ID NO: 12), 200 μM of dNTPs, 1 unit of Taq polymerase, and 0.1 ngof Lambda DNA. Amplification was performed in BioRad CFX96 Real TimeSystem using the following thermal cycling profile: one cycle at 94° C.for 2 min, followed by 50 cycles at 94° C. for 15 seconds and 66° C. for1 minute 20 seconds and “read” cycle for 10 seconds at 60° C. Somereactions were also supplied with 0.2 units of Vent (exo-) polymerase.

Results:

Averaged amplification real-time PCR curves are shown on FIG. 18. Theassay with fluorophor-labeled P10-104 and universal quencheroligonucleotide 10-80 generated a strong signal that was significantlyimproved by adding Vent (exo-) polymerase.

Conclusions:

qPCR assay with fluorophor-labeled P10-104 and a universal quencherrepresent a novel qPCR tool for diagnostic applications. It is lessexpensive for designing multiple assays than the method described inExample 4 because of the use of a universal quencher molecule.

Example 6

KRAS G12V Mutation Assay with SybrGreen Dye.

Materials:

0.1×TE buffer: 10 mM Tris, 0.1 mM EDTA pH=8.0

Genomic DNA isolation kit: Qiagen DNeasy Blood & Tissue Kit #69504

Wild Type human genomic DNA template: Promega human genomic DNA # G1471

Mutant template: (G12V) genomic DNA isolated from freshly harvestedSW480 colorectal adenocarcinoma cells (ATCC # CCL-228)

Oligonucleotides:

has conventional forward primer 10-53 (SEQ ID NO: 6),

has G12V conventional reverse primer 10-48 (SEQ ID NO: 5),

has P10-56 (SEQ ID NO: 8) (i.e., “first polynucleotide”),

has F10-54 (SEQ ID NO: 7) (i.e., “second polynucleotide”),

Real time SYBR Green qPCR mix: BioRad IQ SYBR Green Supermix #170-8882

Methods:

Genomic DNA isolation: Kras G12V human genomic DNA was isolated fromfreshly harvested SW480 cells using Qiagen DNeasy Blood & Tissue Kitaccording to manufacturer protocol, resuspended in 0.1×TE buffer at aconcentration of 100 ng/μ1, aliquoted and stored at −20° C. until use.

Template preparation: To generate genomic DNA templates for real timePCR reactions, Promega WT genomic DNA was spiked with the designatedamount of has G12V genomic DNA so that number of mutant has copiesvaried from one to 14,000 copies per 50 ng of total DNA, then templateDNA was aliquoted and stored at −20° C. until use.

Real time amplification reaction: Amplification was carried out intriplicate in 25 μl aliquots consisting of 12.5 μl of 2× BioRad IQ SYBRGreen Supermix, 200 nM of forward primer 10-53 (SEQ ID NO: 6), 200 nM ofconventional reverse primer 10-48 (SEQ ID NO: 5) for conventional PCRreactions or 200 nM of has P10-56 (SEQ ID NO: 8) and 400 nM of hasF10-54 (SEQ ID NO: 7) for Primer combination amplification reactions and50 ng of template DNA using BioRad CFX96 Real Time System. Amplificationwas performed using the following thermal cycling profile: one cycle at94° C. for 3 minutes, followed by 60 cycles at 94° C. for 15 seconds and66° C. for 1 minute 20 seconds.

Results:

Averaged Primer Combination qPCR curves for normal 50 ng DNA samplescontaining 50% (7,000 mutant DNA copies), 10% (1,400 mutant DNA copies),1% (140 mutant DNA copies), 0.1% (14 mutant DNA copies), 0.01% (1 mutantDNA copy), and 0% of the mutant allele G12V are shown in FIG. 19a . FIG.19b shows analysis of the same DNA samples using conventional primers.In both cases, primers were designed to discriminate the mismatch by thebase located at the 3′ end of has P10-56 or conventional primer.

Conclusions:

Primer Combination KRAS G12V mutation assay was able to detect a singlecopy mutant allele present in the mixture of 14,000 normal DNA sequences(0.01%), while the assay with conventional primers was limited todetection of 140 copies of mutant DNA (1%). There was a 100-foldimprovement in sensitivity when Primer Combinations were used for raremutation detection as compared to conventional primers. Signaloriginating from a single mutant allele can be discriminated from thebackground (2-3 cycle difference).

Example 7

Detection of Mutations Using Primer Combinations and SybrGreen withFixed Samples.

Materials:

WT has HT29 colorectal adenocarcinoma cells ATCC # HTB-38

G12V genomic DNA was isolated from freshly harvested SW480 colorectaladenocarcinoma cells (ATCC # CCL-228).

Methods:

Cells fixation: HT29 cells (the source of has WT DNA) or SW480 cells(source of has G12V DNA) were trypsinized, washed 3 times in ice coldPBS, fixed in 4% formaldehyde for 10 minutes at room temperature, andwashed again 4 times with PBS. After final wash, cell pellets were usedto isolate DNA according to the protocol described in Example 5.

Template preparation and DNA amplification: same as described in Example5.

Results:

Averaged Primer Combination qPCR curves for fixed DNA samples containing100% (14,000 mutant DNA copies), 50% (7,000 mutant DNA copies), 10%(1,400 mutant DNA copies), 1% (140 mutant DNA copies), 0.1% (14 mutantDNA copies), 0.01% (1 mutant DNA copy), and 0% of mutant allele G12V areshown in FIG. 20. Similar to results with non-fixed samples thedetection limit was 0.01% or 1 mutant DNA molecule.

Conclusions:

Primer Combination KRAS G12V mutation qPCR assay sensitivity andselectivity was not affected by cell fixation, indicating the utility ofthis assay for real clinical samples. Signal originating from a singlemutant allele can be discriminated from the background (3 cycledifference).

Example 8

KRAS G12V Assay with a Primer Combination and a Probe Polynucleotide.

Materials:

Template DNA:

Wild Type human genomic DNA from Promega # G1471, mutant G12V DNAisolated from SW480 cells (see Example 6).

Oligos:

has conventional forward primer 10-178 (SEQ ID NO: 16),

has G12V P10-171 (SEQ ID NO: 14) (i.e., “first polynucleotide”),

has F10-174 (SEQ ID NO: 15) (i.e., “second polynucleotide”),

has specific Zen double quenched probe 10-185 (SEQ ID NO: 19) (i.e.,“probe polynucleotide comprising a label and a quencher”).

Real time qPCR mix: BioRad IQ Supermix #170-8862.

Methods:

Real-time amplification reaction: Amplification was carried out intriplicate with 25 μl aliquots consisting of 12.5 μl of 2× BioRad IQSupermix, 200 nM of forward primer 10-178 (SEQ ID NO: 16), 200 nM of hasG12V P10-171 (SEQ ID NO: 14), 400 nM of has F10-174 (SEQ ID NO: 15), 250nM of probe 10-185 (SEQ ID NO: 19) and 50 ng of template DNA usingBioRad CFX96 Real Time System. Amplification was performed using thefollowing thermal cycling profile: one cycle at 94° C. for 3 minutes,followed by 60 cycles at 94° C. for 10 seconds and 66.5° C. for 1 minute20 seconds.

Results:

Averaged Primer Combination qPCR curves for DNA samples containing 100%(14,000 mutant DNA copies), 50% (7,000 mutant DNA copies), 10% (1,400mutant DNA copies), 1% (140 mutant DNA copies), 0.1% (14 mutant DNAcopies), 0.01% (1 mutant DNA copy), and 0% of mutant allele G12V areshown in FIG. 21. Similar to results described in Examples 6 and 7 thedetection limit using the probe polynucleotide was 0.01% or 1 mutant DNAmolecule. Signal intensity decreased with low amounts of mutant DNA(0.1% and 0.01%).

Conclusions:

Primer combination KRAS G12V mutation qPCR assay sensitivity did notimprove significantly from using the probe polynucleotide. Signaloriginating from a single mutant allele can be discriminated from thebackground (flat line up to 65 cycles), showing better selectivity inthe probe polynucleotide-containing assay.

Example 9

KRAS G12V Assay with a Primer Combination, Probe Polynucleotide andBlocker Polynucleotide.

Materials:

Template DNA:

Wild Type human genomic DNA from Promega # G1471, mutant G12V DNAisolated from SW480 cells (see Example 6).

Oligos:

kras P10-184 (SEQ ID NO: 18) (i.e., “first polynucleotide”),

has F10-182 (SEQ ID NO: 17) (i.e., “second polynucleotide”),

has specific Zen double quenched probe 10-210 (SEQ ID NO: 21) (i.e.,“probe polynucleotide”),

has conventional reverse primer 10-208 (SEQ ID NO: 20), (i.e., “reverseprimer”)

blocking oligo 10-213 (SEQ ID NO: 22) (i.e., “blocker polynucleotide”)

Real time qPCR mix: BioRad IQ Supermix #170-8862 Methods:

Real time amplification reaction: Amplifications were carried out intriplicate in 25 μl aliquots consisting of 12.5 μl of 2× BioRad IQSupermix, 200 nM of has P10-184 (SEQ ID NO: 18), 50 nM of has F10-182(SEQ ID NO: 17), 200 nM of reverse primer 10-208 (SEQ ID NO: 20), 250 nMof probe polynucleotide 10-210 (SEQ ID NO: 21), 2000 nM of blockingoligo 10-213 (SEQ ID NO: 22) and 50 ng of template DNA using BioRadCFX96 Real Time System. Amplification was performed using the followingthermal cycling profile: one cycle at 94° C. for 3 minutes, followed by60 cycles at 94° C. for 10 seconds and 65° C. for 1 minute.

Results:

Averaged Primer Combination qPCR curves for DNA samples containing 100%(14,000 mutant DNA copies), 50% (7,000 mutant DNA copies), 10% (1,400mutant DNA copies), 1% (140 mutant DNA copies), 0.1% (14 mutant DNAcopies), 0.01% (1 mutant DNA copy), and 0% of mutant allele G12V areshown in FIG. 22.

Conclusions:

Addition of the blocker oligonucleotide 10-213 (SEQ ID NO: 22) (incombination with the probe polynucleotide) significantly improved thecharacteristics of the assay. Similar to results described in Examples4, 5 and 6 the detection limit using TaqMan probe and the blocker was0.01%, or 1 mutant DNA molecule, but there was ˜100-1000× improvement inselectivity for detection of a single mutant allele than the assaydescribed in Example 6 (5-7 cycles difference between single mutant copyand the background originating from 14,000 normal DNA molecules).

Example 10

Primer Combination KRAS G12V Assay with Probe Polynucleotide, BlockerPolynucleotide, and 3′-Base LNA Modification.

Materials:

Template DNA:

Wild Type human genomic DNA from Promega # G1471, mutant G12V DNAisolated from SW480 cells (see Example 6).

Oligos:

has P10-236 with 3′ LNA (SEQ ID NO: 23) (i.e., “first polynucleotidecomprising a locked nucleic acid at the 3′ end”),

has F10-182 (SEQ ID NO: 17) (i.e., “second polynucleotide”),

has specific Zen double quenched probe 10-210 (SEQ ID NO: 21) (i.e.,“probe polynucleotide”),

has conventional reverse primer 10-208 (SEQ ID NO: 20) (i.e., “reverseprimer”),

blocking oligo 10-213 (SEQ ID NO: 22) (i.e., “blocker polynucleotide”)

Real time qPCR mix: BioRad IQ Supermix #170-8862

Methods:

Real time amplification reaction: Amplifications were carried out intriplicates in 25 μl volume consisting of 12.5 μl of 2×BioRad IQSupermix, 200 nM of kras P10-236 (SEQ ID NO: 23), 50 nM of kras F10-182(SEQ ID NO: 17), 200 nM of reverse primer 10-208 (SEQ ID NO: 20), 250 nMof probe polynucleotide 10-210 (SEQ ID NO: 21), 2000 nM of blockingoligo 10-213 (SEQ ID NO: 22) and 50 ng of template DNA using BioRadCFX96 Real Time System. Amplification was performed using the followingthermal cycling profile: one cycle at 94° C. for 3 minutes, followed by60 cycles at 94° C. for 10 seconds and 65° C. for 1 minute.

Results:

Averaged Primer Combination qPCR curves for DNA samples containing 1%(140 mutant DNA copies) and 0% of mutant allele G12V are shown in FIG.23.

Conclusions:

Primer Combination KRAS g12V assay with probe polynucleotide, blockerpolynucleotide and 3′-base LNA modification of P10-236 demonstrated thebest sensitivity (single mutant allele) and the best selectivity (nosignal from 14,000 wild type DNA molecules) as compared to the previousexamples.

Example 11

Single Mutation Detection Using Improved KRAS G12V Assay at 0.5 Copy ofG12V DNA Per 14,000 copies of WT DNA: analysis of 16 samples

Materials:

Same as Example 10.

Methods:

Template preparation:

To generate 0.5 copy KRAS G12V DNA template, previously isolated SW480genomic DNA was diluted in 10 ng/μl Promega Human genomic DNA to thefinal concentration of 1 SW480 DNA molecule per 10 μl of WT DNA.

Real time amplification reaction:

Amplifications were performed in 16 aliquots with 5 μl of 0.5 copy KRASG12V DNA template or WT DNA using amplification mix composition andprotocol described in Example 10.

Results:

16 replicate experiments using improved KRAS G12V qPCR mutation assayfrom Example 10 and samples containing (statistically) 0.5 copy of G12VDNA per 14,000 copies of WT DNA are shown in FIG. 24 a. 50-60% (8-10 outof 16) of spiked and diluted DNA samples indicated the presence of asingle mutant DNA, and 40-50% (6-8 out of 16) of spiked and diluted DNAsamples showed no signal, in agreement with the statisticalexpectations.

16 replicate experiments using improved KRAS G12V qPCR mutation assayfrom Example 10 and samples containing 14,000 copies of WT DNA (nomutant DNA added) are shown in FIG. 24 b. 94% of WT DNA samples (15 outof 16) showed no signal, indicating a very high selectivity of singlemutant DNA detection by the improved KRAS G12V Primer Combination qPCRmutation assay.

Conclusions:

Primer Combination G12V assay with probe polynucleotide, blocker and3′-modified LNA base had 100% sensitivity and 94-100% selectivity fordetection of a single mutant allele in excess of more than 10,000non-mutant DNA molecules. Such parameters of the G12V assay satisfy themost demanding characteristics of a diagnostic assay. The assay isideally suitable for detection of rare cancer cells circulating in bloodfor efficient and non-invasive management of CRC and NSCLC patients.

1. A polynucleotide primer combination comprising a first polynucleotideand a second polynucleotide, the first polynucleotide (P) comprising afirst domain (Pa) having a sequence that is complementary to a firsttarget polynucleotide region (TO and a second domain (Pc) comprising aunique polynucleotide sequence, and the second polynucleotide (F)comprising a first domain (Fb) having a sequence that is complementaryto a second target polynucleotide region (T₂) and a second domain (Fd)comprising a polynucleotide sequence sufficiently complementary to Pcsuch that Pc and Fd will hybridize under appropriate conditions, whereinthe target polynucleotide has a secondary structure that is denatured byhybridization of Fb to the target polynucleotide.
 2. The polynucleotideprimer combination of claim 1 wherein the secondary structure of thetarget polynucleotide inhibits polymerase extension of the targetpolynucleotide in the absence of F.
 3. The polynucleotide primercombination of claim 1 wherein the P and/or F further comprise amodified nucleic acid.
 4. (canceled)
 5. A polynucleotide primercombination comprising a first polynucleotide, a second polynucleotide,and a blocker polynucleotide, the first polynucleotide (P) comprising afirst domain (Pa) having a sequence that is complementary to a firsttarget polynucleotide region (TO and a second domain (Pc) comprising aunique polynucleotide sequence, the second polynucleotide (F) comprisinga first domain (Fb) having a sequence that is complementary to a secondtarget polynucleotide region (T₂) and a second domain (Fd) comprising apolynucleotide sequence sufficiently complementary to Pc such that Pcand Fd will hybridize under appropriate conditions, and the blockerpolynucleotide comprising a nucleotide sequence that is complementary toa third target polynucleotide region (T₃), wherein T₃ is located 5′ ofT₁ and T₂.
 6. The polynucleotide primer combination of claim 5 wherein anucleotide at the 3′ end of P and a nucleotide at the 5′ end of theblocker polynucleotide overlap.
 7. The polynucleotide primer combinationof claim 6 wherein the blocker polynucleotide has a sequence thatoverlaps Pa over the whole length of Pa.
 8. The polynucleotide primercombination of claim 6 wherein the nucleotide at the 3′ end of P and thenucleotide at the 5′ end of the blocker polynucleotide are different. 9.(canceled)
 10. (canceled)
 11. (canceled)
 12. (canceled)
 13. (canceled)14. (canceled)
 15. (canceled)
 16. (canceled)
 17. (canceled) 18.(canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)23. (canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled) 27.(canceled)
 28. (canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled)32. The polynucleotide primer combination of claim 1 wherein Pa is fromabout 5 bases in length to about 30 bases in length, about 5 bases inlength to about 20 bases in length, about 5 bases in length to about 15bases in length, about 5 bases in length to about 10 bases in length,about 5 bases in length to about 8 bases in length.
 33. Thepolynucleotide primer combination of claim 1 wherein Pc is from about 5bases in length to about 200 bases in length, about 5 bases in length toabout 150 bases in length, about 5 bases in length to about 100 bases inlength, about 5 bases in length to about 50 bases in length, about 5bases in length to about 45 bases in length, about 5 bases in length toabout 40 bases in length, about 5 bases in length to about 35 bases inlength, about 5 bases in length to about 30 bases in length, about 5bases in length to about 25 bases in length, about 5 bases in length toabout 20 bases in length, about 5 bases in length to about 15 bases inlength, about 10 to about 50 bases in length, about 10 bases in lengthto about 45 bases in length, about 10 bases in length to about 40 basesin length, about 10 bases in length to about 35 bases in length, about10 bases in length to about 30 bases in length, about 10 bases in lengthto about 25 bases in length, about 10 bases in length to about 20 basesin length, or about 10 bases in length to about 15 bases in length. 34.The polynucleotide primer combination of claim 1 wherein Fb is fromabout 10 bases in length to about 5000 bases in length, about 10 basesin length to about 4000 bases in length, about 10 bases in length toabout 3000 bases in length, about 10 bases in length to about 2000 basesin length, about 10 bases in length to about 1000 bases in length, about10 bases in length to about 500 bases in length, about 10 bases inlength to about 250 bases in length, about 10 bases in length to about200 bases in length, about 10 bases in length to about 150 bases inlength, about 10 bases in length to about 100 bases in length, about 10bases in length to about 95 bases in length, about 10 bases in length toabout 90 bases in length, about 10 bases in length to about 85 bases inlength, about 10 bases in length to about 80 bases in length, about 10bases in length to about 75 bases in length, about 10 bases in length toabout 70 bases in length, about 10 bases in length to about 65 bases inlength, about 10 bases in length to about 60 bases in length, about 10bases in length to about 55 bases in length, about 10 bases in length toabout 50 bases in length, about 10 bases in length to about 45 bases inlength, about 10 bases in length to about 40 bases in length, about 10bases in length to about 35 bases in length, about 10 bases in length toabout 30 bases in length, or about 10 bases in length to about 100 basesin length.
 35. The polynucleotide primer combination of claim 1 whereinFd is from about 5 bases in length to about 200 bases in length, about 5bases in length to about 150 bases in length, about 5 bases in length toabout 100 bases in length, about 5 bases in length to about 50 bases inlength, about 5 bases in length to about 45 bases in length, about 5bases in length to about 40 bases in length, about 5 bases in length toabout 35 bases in length, about 5 bases in length to about 30 bases inlength, about 5 bases in length to about 25 bases in length, about 5bases in length to about 20 bases in length, about 5 bases in length toabout 15 bases in length, about 10 to about 50 bases in length, about 10bases in length to about 45 bases in length, about 10 bases in length toabout 40 bases in length, about 10 bases in length to about 35 bases inlength, about 10 bases in length to about 30 bases in length, about 10bases in length to about 25 bases in length, about 10 bases in length toabout 20 bases in length, or about 10 bases in length to about 15 basesin length.
 36. (canceled)
 37. (canceled)
 38. (canceled)
 39. (canceled)40. (canceled)
 41. (canceled)
 42. (canceled)
 43. (canceled) 44.(canceled)
 45. A method of detecting the presence of a targetpolynucleotide in a sample with a primer combination, the primercombination comprising a first polynucleotide and a secondpolynucleotide, the first polynucleotide (P) comprising a first domain(Pa) having a sequence that is fully complementary to a first targetpolynucleotide region (T₁) and a second domain (Pc) comprising a uniquepolynucleotide sequence, Pa having a sequence that is not fullycomplementary to a non-target polynucleotide in the sample and thesecond polynucleotide (F) comprising a first domain (Fb) that iscomplementary to a second target polynucleotide region (T₂) and a seconddomain (Fd) comprising a polynucleotide sequence sufficientlycomplementary to Pc such that Pc and Fd will hybridize under appropriateconditions, the method comprising the steps of: contacting the samplewith the primer combination and a polymerase under conditions that allowextension of a sequence from Pa which is complementary to the targetpolynucleotide when the target polynucleotide is present in the sampleand detecting the sequence extended from Pa indicating the presence ofthe target polynucleotide in the sample.
 46. The method of claim 45wherein the method provides a change in sequence detection from a samplewith a non-target polynucleotide compared to sequence detection from asample with a target polynucleotide.
 47. (canceled)
 48. (canceled) 49.(canceled)
 50. (canceled)
 51. (canceled)
 52. The method of claim 45wherein detection is carried out in real time.
 53. (canceled) 54.(canceled)
 55. (canceled)
 56. (canceled)
 57. (canceled)
 58. (canceled)59. (canceled)
 60. (canceled)
 61. (canceled)
 62. (canceled) 63.(canceled)
 64. (canceled)
 65. (canceled)
 66. (canceled)
 67. (canceled)68. (canceled)
 69. (canceled)